Multipotent stem cells and uses thereof

ABSTRACT

The invention provides a quiescent stem cell having the capacity to differentiate into ectoderm, mesoderm and endoderm, and which does not express cell surface markers including MHC class I, MHC class II, CD44, CD45, CD13, CD34, CD49c, CD73, CD105, CD90, CD66A, CD66E, CXCR4, CD133 or an SSEA. The invention further provides a proliferative stem cell, which expresses genes including Oct-4, Nanog, Sox2, GDF3, P16INK4, BMI, Notch, HDAC4, TERT, Rex-1, TWIST, KLF-4 and Stella but does not express cell surface markers including MHC class I, MHC class II, CD44, CD45, CD13, CD34, CD49c, CD73, CD105, CD90, CD66A, CD66E, CXCF4, CD133 or an SSEA. The cells of the invention can be isolated from adult mammals, have embryonic cell characteristics, and can form embryoid bodies. Methods for obtaining the stem cells, as well as methods of treating diseases and differentiating the stem cells, are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a Continuation Application of U.S. application Ser.No. 13/799,849, filed Mar. 13, 2013, which is a Continuation applicationof Ser. No. 12/598,047, filed Oct. 29, 2009, which is a 35 U.S.C. § 371National Phase Entry Application of International Application No.PCT/US2008/005742, filed May 5, 2008, which designates the U.S., andwhich claims benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 60/927,596, filed May 3, 2007, the contents of each ofwhich are incorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

This work was supported by NIAMS grant AR050243. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

A stem cell is commonly defined as a cell that (i) is capable ofrenewing itself; and (ii) can give rise to more than one type of cellthrough asymmetric cell division (Watt et al., Science, 284:1427-1430,2000). Stem cells typically give rise to a type of multipotent cellcalled a progenitor cell; progenitor cells, in turn, proliferate anddifferentiate into lineage-committed cells that populate the body.

Pluripotent stem cells are thought to have the potential todifferentiate into almost any cell type, while multipotent stem cellsare believed to have the potential to differentiate into many cell types(Robertson, Meth. Cell Biol. 75:173, 1997; Pedersen, Reprod. Fertil.Dev. 6:543-552, 1994).

Stem cells exist in many tissues of embryos and adult mammals. Manydifferent types of mammalian stem cells have been characterized andcertain stem cells have not only been isolated and characterized, buthave also been cultured under conditions to allow differentiation to alimited extent. Both adult and embryonic stem cells are able todifferentiate into a variety of cell types and, accordingly, may be asource of replacement cells and tissues that are damaged in the courseof disease or infection, or because of congenital abnormalities(Lovell-Badge, Nature 414:88-91, 2001; Donovan et al., Nature 414:92-97,2001). Various types of putative stem cells exist which, whendifferentiated into mature cells, carry out the unique functions ofparticular tissues, such as heart, liver, or neuronal tissue. Thesecells are important for the treatment of a wide variety of disorders,including malignancies, inborn effors of metabolism, hemoglobinopathies,immunodeficiencies and the replacement of damaged and diseased tissues.Recent success at transplanting such stem cells have provided newclinical tools to reconstitute and/or supplement bone marrow aftermyeloablation due to disease, exposure to toxic chemical and/orradiation. Further evidence exists that demonstrates that stem cells canbe employed to repopulate many, if not all, tissues and restorephysiologic and anatomic functionality. The application of stem cells intissue engineering, gene therapy delivery and cell therapeutics is alsoadvancing rapidly.

Prior to the present invention, a basic problem existed, i.e., thatobtaining sufficient quantities and populations of human stem cellswhich are capable of differentiating into most cell types was nearlyimpossible. Stem cells are in critically short supply.

Obtaining sufficient numbers of human stem cells has been problematicfor several reasons. First, isolation of normally occurring populationsof stem cells in adult tissues has been technically difficult and costlydue, in part, to very limited quantity found in blood or tissue. Theisolation of the cells is generally laborious, involving the harvestingof cells or tissues from a patient or donor, culturing and/orpropagating the cells in vitro. Certain cell types, such as nerve cellsand cardiac cells, differentiate during development, and adult organismsare not known to generally replace these cells. Even in cell types thatare replaced in adult organisms (e.g., epithelial cells andhematopoietic cells), it has been a significant challenge to readily andinexpensively obtain stem cells in significant quantities. For example,mammalian hematopoietic cells (e.g., lymphoid, myeloid, and erythroidcells) are all believed to be generated by a single cell type called thehematopoietic “stem cell” (Civin et al., J. Immunol. 133:157-165, 1984).However, these hematopoietic stem cells are very rare in adults,accounting for approximately 0.01% of bone marrow cells. Isolation ofthese cells based on surface proteins such as CD34 results in very lowyields. Schemes to fractionate human hematopoietic cells into lineagecommitted and non-committed progenitors are technically complicated andoften do not permit the recovery of a sufficient number of cells toaddress multilineage differentiation (Berenson et al., 1991; Terstappenet al., 1991; Brandt et al., J. Clin. Invest. 82:1017-1027, 1988;Landsdorp et al., J. Exp. Med. 175:1501-1509, 1992; Baum et al., Proc.Natl. Acad. Sci. USA 89:2804-2808, 1992)

A second reason that obtaining sufficient number of human stem cells hasbeen problematic is that procurement of these cells from embryos orfetal tissue has raised religious, ethical, and legal concerns.Alternative sources that do not require the use of cells procured fromembryonic or fetal tissue are therefore highly desirable for clinicaluse of stem cells. Prior to the present invention, there have been fewviable alternative sources of stem cells, particularly human stem cells.

It would therefore be of particularly great value in treating a widevariety of diseases to have an easily accessible quantity of embryoniclike stem cells that are found in the adult body that can reliablydifferentiate into a desired phenotype.

In addition, it would also be advantageous to have stem cells that donot require feeder cells. Many adult stem cell propagation protocolsrequire such cells, which creates risks including infection, cellfusion, and/or contamination. As such, adult stem cells are have oftenbeen very difficult to expand in culture (Reya et al., Nature414:105-111, 2001; Tang et al. Science 291:868-871, 2001).

Thus, there remains a need in the art to develop methods foridentifying, propagating, and altering the state (e.g., bydifferentiation or dedifferentiation) of stem cells and to provide asource of cells that are transplantable to in vivo tissues in order toreplace damaged or diseased tissue.

SUMMARY OF THE INVENTION

A purified population of stem cells that exist in the synovial fluid andblood is disclosed. These cells are embryonic in character and prior toculture do not present the surface markers generally associated withother adult stem cells, even after days in culture. These cells alsoexpress key embryonic transcription factors within a few days ofisolation, in contrast to other adult stem cells, which require longerperiods of culture before such expression. Further, these cell candifferentiate into all three germ layers (mesoderm, ectoderm, andendoderm) and do not form teratoma bodies in vitro. This discoveryallows for a non-controversial supply of easily attainableembryonic-like stem cells.

Accordingly, in one aspect, the invention features an isolated (e.g.,purified or substantially purified) stem cell which is capable ofproliferating and differentiating into ectoderm, mesoderm, and endoderm,expresses at least one of (e.g., at least 2, 4, 5, or 6 of) Oct-4,Nanog, Sox-2, KLF4, c-Myc, Rex-1, GDF-3, LIF receptor, and Stella, anddoes not express at least one of (e.g., at least 2, 3, 4, 5, 6, 7, 8, orall of) MEC class I, MHC class II, CD44, CD45, CD13, CD34, CD49c, CD66b,CD73, CD105, and CD90 cell surface proteins.

In another aspect, the invention features an isolated (e.g., purified orsubstantially purified) quiescent stem cell which is capable ofproliferating and differentiating into ectoderm, mesoderm, and endodermand does not express Oct-4 and at least one of (e.g., at least 2, 3, 4,5, 6, 7, 8, or all of) MHC class I, MEC class II, CD44, CD45, CD13,CD34, CD49c, CD66b, CD73, CD105, and CD90 cell surface proteins. Thestem cell may be proliferative following 1, 2, 3, 4, 5, 6, 7, 8, or 10days in culture.

In either of the above two aspects, the stem cell may not express CD13,CD44, CD45, CD90, and CD105. The stem cell may be isolated from synovialfluid, blood, or other tissue. The cell of may be isolated from a humanor non-human animal (e.g., any described herein). The cell may be about6 μm to about 15 μm or to 20 μm in size. The cell may further contain aheterologous nucleic acid sequence, which may include a stemcell-specific promoter (e.g., an embryonic transcription factor promotersuch as an Oct-4 promoter, Nanog promoter, Sox-2 promoter, KLF4promoter, c-Myc promoter, Rex-1 promoter, GDF-3 promoter, Stellapromoter, FoxD3 promoter, Polycomb Repressor Complex 2 promoter, andCTCF promoter). The promoter may be operably linked to a detectable geneproduct (e.g., a fluorescent protein such as a GFP) or any gene product,including those described herein.

In another aspect the invention features a population of cells includingcells of either of the previous two aspects. The population may containat least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% ofthe cells of the previous two aspects (e.g., an embryoid body). Thepopulation may further be part of a composition including acryoprotectant (e.g., any described herein). The population may bepresent with a dendritic cell or an antigen presenting cell (e.g., in acell culture including a dendritic cell or antigen-presenting cell). Inanother embodiment, the population of cells may be part of a compositionwith other types of stem cells (e.g., any known in the art such asmesenchymal cells or embryonic stem cells) or differentiated cells(e.g., any described herein, including myocytes, adipocytes,fibromyoblasts, ectodermal cells, muscle cells, osteoblasts,chondrocytes, endothelial cells, fibroblasts, pancreatic cells,hepatocytes, bile duct cells, bone marrow cells, neural cells, andgenitourinary cells. Such cells may be autologous or allogenic to thestem cells of the invention.

In another aspect, the invention features a method for isolating apopulation of stem cells. The method includes the steps (a) providing abodily fluid from a subject (e.g., a mammal such as a human); (b)enriching for a population of cells that are about 6 μm to 20 μm insize; and may optionally include (c) depleting cells from the populationexpressing stem cell surface markers or MHC proteins (e.g., anydescribed herein), thereby isolating a population of stem cells. Step(c) may include depleting cells expressing MHC class I, CD66b,glycophorin a, or glyclophorin b, or any of the cell surface markersdescribed herein. The subject may be administered a stem cell mobilizingagent prior to step (a) providing. The subject may be suffering fromosteoarthritis. The method may further include (d) cryopreserving thecells. In another embodiment, the method further includes (d)transfecting the cells with a polynucleotide vector containing a stemcell-specific promoter (e.g., an Oct-4, Nanog, Sox-9, GDF3, Rex-1, orSox-2 promoter, or any promoter described herein) operably linked to areporter or selection gene; and (e) further enriching the population forthe stem cells using expression of the reporter or selection gene (e.g.,using flow cytometry). In another embodiment, the method furtherincludes (d) contacting the cells with a detectable compound (e.g.,carboxyfluorescein diacetate, succinimidyl ester, or Aldefluor) whichenters said cells, the compound being selectively detectable inproliferating and non-proliferating cells; and (e) enriching thepopulation of cells for the proliferating cells. In another embodiment,the method further includes culturing the cells under conditions whichform embryoid bodies (e.g., those described herein).

The method may further include separating (e.g., by cell depletion) celltypes such as granulocytes, T-cells, B-cells, NK-cell, red blood cells,or any combination thereof, from the stem cells of the invention. Themethod may further include culturing the population of stem cell underconditions which support proliferation of the cells (e.g., where theculturing conditions include the presence of dendritic cells orantigen-presenting cells). In any of the embodiments of this aspect ofthe invention, the cells may further be cryopreserved.

The invention also features a cell produced by any of the above methods.The invention also provides a method for identifying a stem cell, themethod including the steps of introducing into a stem cell a vectorcomprising a stem cell-specific promoter coupled to at least oneselectable marker gene, wherein said stem cell that does not express atleast one of (e.g., at least 2, 3, 4, 5, 6, 7, 8, or all of) MT-IC classI, MHC class II, CD44, CD45, CD13, CD34, CD49c, CD66b, CD73, CD105, andCD90 cell surface proteins, is capable of differentiating into mesoderm,ectoderm, and endoderm; and expresses at least one embryonictranscription factor (e.g., Oct-4, Nanog, Sox-2, Rex-1, GDF-3, Stella,FoxD3, Polycomb), expressing the selectable marker gene from thestem-cell specific promoter in said stem cell; and detecting expressionof the marker gene in the stem cell, thereby identifying the stem cell.The method may further comprise isolating the stem cell. The stem cellcan be derived from the bodily fluid of a mammal, such as synovial fluidor blood. Preferably, the mammal is a human. In certain embodiments, thecell does not express CD13, CD44, and CD90.

In various embodiments, the stem cell-specific promoter is an Oct-4promoter, Nanog promoter, Sox-2 promoter, Rex-1 promoter, GDF-3promoter, Stella promoter, FoxD3 promoter, Polycomb Repressor Complex 2promoter, or CTCF promoter. In one embodiment, the stem cell-specificpromoter is flanked by loxP sites. In another embodiment, the vector isa retroviral vector.

In yet another embodiment, the selectable marker gene encodes afluorescent protein, such as a Green Fluorescent Protein (GFP).

In yet another embodiment, the vector comprises two selectable markergenes. In a specific embodiment, the two selectable marker genes are afluorescent protein and a protein sensitive to drug selection.

In yet another embodiment, the selectable marker gene encodes a cellsurface protein.

In another aspect, the invention provides a stem cell isolated by amethod comprising the steps of introducing into a stem cell a vectorcomprising a stem cell-specific promoter coupled to at least oneselectable marker gene, wherein said stem cell does not express MHCclass I, MHC class II, CD44, CD45, CD13, CD34, CD49c, CD73, CD105 andCD90 cell surface proteins; expressing the selectable marker gene fromthe stem-cell specific promoter in said stem cell; and detectingexpression of the marker gene in the stem cell.

In yet another aspect, the invention provides a method for isolatingproliferative stem cells from a population of mobilized cells, themethod comprising the steps of introducing into a population ofmobilized cells a vector comprising a stem cell-specific promotercoupled to at least one selectable marker gene, wherein said populationcomprises proliferative stem cells which express Oct-4, Nanog, Sox-2,Rex-1, GDF-3, and Stella, and do not express MHC class I, MHC class II,CD44, CD45, CD13, CD34, CD49c, CD73, CD105 and CD90 cell surfacemarkers; expressing the selectable marker gene in said proliferativestem cells; detecting expression of the marker gene in saidproliferative stem cells; and isolating said proliferative stem cellsfrom the population of mobilized cells.

In one embodiment, about 5% to about 30% proliferative stem cells areisolated from a population of about 500,000-12,000,000 mobilized cellswithout expansion in vitro.

In yet another aspect, the invention provides proliferative stem cellsisolated by the method comprising the steps of introducing into apopulation of mobilized cells a vector comprising a stem cell-specificpromoter coupled to at least one selectable marker gene, wherein saidpopulation comprises proliferative stem cells which express Oct-4,Nanog, Sox-2, Rex-1, GDF-3 and Stella, and do not express MEC class I,MHC class II, CD44, CD45, CD13, CD34, CD49c, CD73, CD105 and CD90 cellsurface markers; expressing the selectable marker gene in saidproliferative stem cells; detecting expression of the marker gene insaid proliferative stem cells.

In yet another aspect, the invention provides a method of obtaining stemcells from synovial fluid, the method comprising the steps of obtainingsynovial fluid from a subject optionally treated with a stem cellmobilizing agent; centrifuging the synovial fluid at about 200 g to forma first pellet of cells, thereby obtaining a population of stem cells.The method may further include any of the enrichment proceduresdescribed herein. Such methods include applying said cells to adiscontinuous density gradient and centrifuging the gradient at about500 g to form a second pellet of cells; suspending the second pellet ofcells in a solution to form a suspension. The method may also includecontacting the suspension with an agent that binds to an MHC class Icell surface protein; to form a binding complex between said agent andcells that express an MEC class I cell surface protein; contacting thesuspension with an agent that binds to glycophorin to form a bindingcomplex between said agent and cells that express glycophorin; andremoving the binding complex from the suspension to form an MHC class Iand glycophorin-depleted suspension, thereby obtaining stem cells fromsynovial fluid. The method may further comprise isolating proliferativestem cells from the obtained stem cells, wherein said proliferative stemcells express Oct-4, Nanog, Sox-2, Rex-1, GDF-3 and Stella, and do notexpress MHC class I, MHC class II, CD44, CD45, CD13, CD34, CD49c, CD73,CD105 and CD90 cell surface markers. The invention also features a stemcell isolated by this method.

In yet another aspect, the invention provides a stem cell obtained fromsynovial fluid by a method comprising the steps of obtaining synovialfluid from a subject optionally treated with a stem cell mobilizingagent; centrifuging the synovial fluid at about 200 g to form a firstpellet of cells; applying said cells to a discontinuous density gradientand centrifuging the gradient at about 500 g to form a second pellet ofcells; suspending the second pellet of cells in a solution to form asuspension; contacting the suspension with an agent that binds to an MHCclass I cell surface protein; to form a binding complex between saidagent and cells that express an MHC class I cell surface protein;contacting the suspension with an agent that binds to glycophorin toform a binding complex between said agent and cells that expressglycophorin; and removing the binding complex from the suspension toform an MHC class I and glycophorin-depleted suspension.

In yet another aspect, the invention provides a method for thedifferentiation of a stem cell of the invention into a cell lineage of agerm layer selected from the group consisting of ectoderm, mesoderm andendoderm, and/or a specific cell type including but not limited to aneural, glial, chondroblast, osteoblast, adipocyte, hepatocyte, musclecell (e.g., smooth muscle or skeletal muscle), cardiac cell, pancreaticcell, pulmonary cell, and endothelial cell.

In yet another aspect, the invention provides a method of forming anadipocyte by culturing a stem cell of any of the previous aspects underadipocyte forming conditions (e.g., with dexamethasone,3-isobutyl-1-methylxanthine (IBMX), insulin and indomethacin), therebyforming an adipocyte.

In yet another aspect, the invention provides a method of forming acartilage cell by culturing a stem cell of any of the previous aspectsunder chrondrocyte-forming conditions (e.g. with TGF-β1 and BMP-4)thereby forming a cartilage cell.

In yet another aspect, the invention provides a method of forming a bonecell by culturing a stem cell of any of the previous aspects underosteoblast-5 forming conditions (e.g. with BMP-2) thereby forming a bonecell.

In yet another aspect, the invention provides a method of forming amuscle cell by culturing a stem cell of any of the previous aspectsunder muscle cell-forming conditions (e.g., with PDGF and TGF-(β1),thereby forming a muscle cell.

In yet another aspect, the invention provides a method of forming aneural cell by culturing a stem cell of any of the previous aspectsunder neural cell-forming conditions (e.g., with bFGF, FGF-8, SHH andBDNF), thereby forming a neuron.

In yet another aspect, the invention provides a method of forming ahepatocyte by contacting a stem cell of any of the previous aspectsunder hepatocyte-forming conditions (e.g., with hepatocyte growth factor(HGF) and FGF-4), thereby forming a hepatocyte.

In yet another aspect, the invention provides a method of forming anendothelial cell by contacting a stem cell of any of the previousaspects under endothelial cell-forming conditions (e.g., with VEGF),thereby forming an endothelial cell.

In yet another aspect, the invention provides a method of forming ahematopoietic cell by culturing a stem cell of any of the previousaspects under hematopoietic cell-forming conditions (e.g., with one ormore of bone morphogenic protein-4 (BMP4), VEGF, bFGF, stem cell factor(SCF), Flt3L, hyper IL6, thrombopoietin (TPO), and erythropoietin(EPO)), thereby forming a hematopoietic cell.

In any of the above differentiating methods, the method may furtherinclude transplanting the differentiated cell into a subject (e.g., ahuman).

The invention also features the use of stem cells to treat disease. Thestem cells of the invention may be used to treat any disease orcondition including but not limited to those described herein.

In another aspect, the invention features a method for promoting woundhealing in a subject. The method includes administering a stem cell ofany of the above aspects, or a committed or differentiated progeny ofthe stem cell, to the wound or to a site near the wound in an amountsufficient to promote the healing of the wound. The administration ofthe cells may result in reduced scarring at the wound site.

In another aspect, the invention features a method for treating acardiovascular disease in a subject. The method includes administeringto the subject a stem cell of any of the above aspects, or a committedor differentiated progeny of the stem cell, in an amount sufficient totreat the disease (e.g., myocardial infarction, congestive heartfailure, ischemic cardiomyopathy, and coronary artery disease).

In another aspect, the invention features a method of increasingvascularization in a subject. The method includes administering to thesubject a stem cell of any of the above aspects, or a committed ordifferentiated progeny of the stem cell, in an amount sufficient toincrease vascularization. For example, the subject may be suffering fromtype II diabetes.

In another aspect, the invention features a method of treating aneurological disorder or neurological damage. The method includesadministering to the subject a stem cell of any of the above aspects, ora committed or differentiated progeny of the stem cell, in an amountsufficient to treat the disorder. The disorder may be aneurodegenerative disease (e.g., Parkinson's disease or anyneurodegenerative disease described herein).

In another aspect, the invention features a method of suppressing animmune response in a subject (e.g., a subject suffering from anautoimmune disease such as those described herein) by administering astem cell of the invention to the subject.

In another aspect, the invention features a method of treating skeletalmuscle disease such as fibrosis (e.g., muscular dystrophy, such as suchas Duchenne's and Becker's muscular dystrophy, and denervation atrophy),by administering a stem cell of the invention, or a differentiatedprogeny of the stem cell, in an amount sufficient to treat the disease.

In another aspect, the invention features a method of treating anautoimmune disease (e.g., any described herein). The method includesadministering to the subject a stem cell of any of the above aspects, ora committed or differentiated progeny of the stem cell, in an amountsufficient to treat the disease.

In another aspect, the invention features a method of replacing orrepairing bone or cartilage. The method includes administering to thesubject a stem cell of any of the above aspects, or a committed ordifferentiated progeny of the stem cell, in an amount sufficient topartially or completely repair or replace the cartilage or bone.

In any of the above treatment methods, the stem cells of the inventionmay be administered simultaneously with, prior to, or followingadministration of a differentiated cell (e.g., an autologous orallogenic cell).

In any of the above treatment methods, autologous or allogenic stem 20cells may be administered.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1a-1e show dot plots of undifferentiated stem cells of theinvention. The freshly isolated stem cells were sorted into 3 groups asshown, Groups A, B, and C, and analyzed for Oct-4, Rex-1, Runx2, Sox-9,Nanog, Class I, CD44, and CD45 expression. FIG. 1a shows forward andside scatter of synovial fluid mononuclear cells. FIG. 1b shows Group Acell surface profile: small size and side scatter. FIG. 1c shows group Bcell surface profile: medium size and small side scatter. FIG. 1d showsgroup C cell surface profile: large size and small side scatter. FIG. 1eshows a comparison of Class-I and CD44 expression in Groups A, B, and C.

FIG. 2 shows the expression of embryonic stem cell genes: Nanog, Oct-4,Rex-1 and Sox-2.

FIG. 3 shows expansion and proliferative capacity of undifferentiatedstem cells of the invention. FIG. 3a depicts a low-power lightmicroscopic view of cell morphology after culture for 3 days, 6 days,and 9 days. In FIG. 3b , freshly isolated stem cells were pulsed withCFSE and the percent positive was assessed after 6 days; the white barregion represents the percentage of highly proliferative stem cells, thestripped bar region represents the percentage of moderatelyproliferative stem cells, and black bar region represents the percentageof nonproliferative stem cells.

FIG. 4 shows differentiation of stem cells into osteoblasts, adipocytes,and a neuron.

FIG. 5 shows expression of osteoblast- or adipocyte-specific genes.

FIG. 6 shows a slide dot plot of the Oct-4 intercellular stain.

FIGS. 7a-7d show an example of stem cells transduced with Lenti-Oct-4GFP (3.5 kb Oct 4 promoter) or Lenti-Nanog-GFP (2.5 kb Nanog promoter).Freshly isolated stem cells were sorted as previously described, pulsedwith 107-108 viral particles per ml, and assessed for fluorescencedaily. FIG. 7a shows a stem cell aggregate 3 days after transduction,low (left panel) and high (right panel). FIG. 7b shows a stem cellaggregate 4 days after transduction, at low (left), medium (middle), andhigh (right) magnifications. FIG. 7c shows a stem cell aggregate 9 daysafter transduction, at low (left) and high (right) magnifications. FIG.7d shows a stem cell aggregate 5 days after transduction, at low (left),medium (middle), and high (right) magnifications.

FIGS. 8a-8c are maps of the lentiviral vector for the stem cellexpression. FIG. 8a shows H2B-EGFP, and FIG. 8b shows GFP-ZEOCIN inhuman adult stem cells. FIG. 8c is a map of the lentiviral vector forthe stem-cell specific expression of a master regulator gene andIRES-EGFP in human adult stem cells.

FIGS. 9a and 9b show co-transducible lentiviral vectors. FIG. 9a shows aco-transducible lentiviral vector system for the tetracycline-inducibleand stem/lineage progenitor cell-specific expression of a masterregulator gene and IRES EYFP in human adult stem cells. FIG. 9b shows aco-transducible lentiviral vector system for the tetracycline-inducibleexpression of a TAT-HA master regulator fusion gene construct and lRESEYPF in human feeder cells or human embryonic stem cells or derivativesof human embryonic stem cells.

FIGS. 10a-10c show lentiviral transduction and FACS (FIG. 10a ) orZEOCIN (FIG. 10b ) selection of human adult stem cells. FIG. 10c showsexpression of master genes in human adult stem cells.

FIGS. 11a and 11b show tetracycline-inducible and stem/lineageprogenitor cell-specific expression of master gene(s) (FIG. 11a ) orTAT-HA-master gene (FIG. 11b ) constructs in human adult stem cellsand/or human adult stem/cell lineage progenitor cells and co-culturewith human adult stem cells.

FIGS. 12a and 12b shows schema for the generation of transgenic mice.FIG. 12a shows generation of humanized rTtA transgenic mice for the invivo expansion of repopulating human cell lineage-specificprogenitor/stem cells. FIG. 12b shows generation of humanized cretransgenic mice for the in vivo expansion of repopulating humancell-lineage specific progenitor/stem cells.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, a “stem cell” is a multipotent or pluripotent cell that(i) is capable of self-renewal; and (ii) can give rise to more than onetype of cell through asymmetric cell division. The term “self renewal”as used herein, refers to the process by which a stem cell divides togenerate one (asymmetric division) or two (symmetric division) daughtercells having development potential indistinguishable from the mothercell. Self renewal involves both proliferation and the maintenance of anundifferentiated state.

As used herein, a “proliferative stem cell” refers to a stem cell thatis 30 rapidly dividing, for example, at a rate of one division every 12,18, 24, 36, or 48 hours.

As used herein, a “quiescent stem cell” refers to a stem cell that isnot dividing and is in the Gap0 (G0) phase of the cell cycle. A“quiescent stem cell” expresses genes responsible for sustaining anepigenetic state of silence, which refers to a state in which thecellular chromatin is organized such that gene expression issubstantially decreased. For example, acetylation of histone H3 and H4correlates with gene expression activation, while deacetylationcorrelates with gene expression silencing (Fry et al., Curr. Biol.11:R185-197, 2001). To date at least eight acetylatable lysine positionsare known in the N termini of histone H3 (K9, K14, K18, and K23) and H4(K5, K8, K12, and K16) and six methylatable lysine positions exist inthose of histone H3 (K4, K9, K27, K36, and K79) and H4 (K20).Methylation of histone H3 (which can be acetylated at lysine positionK4) also marks active chromatin, which contrasts with the modulation ofinactive chromatin by methylation of H3 (which can be methylated at K9)(Lachner et al., J. Cell Sci. 116:2117-2124, 2003). Methylation of H3 atposition K27 is an epigenetic marker for recruitment of polycomb group(Pc-G) complexes (Czermin et al., Cell 111:185-196, 2002) and isprominent in the inactivated X chromosome of female mammalian somaticcells (Plath et al., Science 300:131-135, 2003; Silva et al., Dev. Cell4:481-495, 2003; Cao et al., Science 298:1039-1043, 2002). Thus,acetylation and methylation of histone H3 and H4 amino termini result inregulation of gene activity through the modulation of chromatinconformation, which propagates stably activated or silenced chromatindomains.

As used herein, the term “mobilized cells” refers to cells which havebeen exposed to an agent (e.g., any of those described herein) thatpromotes movement of the cells from the bone marrow into the peripheralblood and/or other reservoirs of the body (e.g., synovial fluid) ortissue.

As used herein, the term “stem cell-specific promoter” is a promoterthat is capable of driving transcription of a gene in a multipotent stemcell, but not in a lineage-committed or differentiated cell. Exemplarystem cell-specific promoters are described herein.

A “nucleic acid molecule” is a strand of linked nucleic acids. The term“nucleic acid” is well known in the art. A “nucleic acid” as used hereinwill generally refer to a molecule (i.e., a strand) of DNA, RNA, or aderivative or analog thereof, comprising a nucleobase. A nucleobaseincludes, for example, a naturally occurring purine or pyrimidine basefound in DNA (e.g., an adenine, guanine, thymine, or cytosine) or RNA(e.g., an adenine, guanine, uracil, or cytosine).

As used herein, the term “heterologous nucleic acid molecule” refers toany heterologous polynucleotide sequence. The sequence can comprise apolynucleotide sequence obtained from a source other than the cell intowhich it is introduced (e.g., an exogenous sequence). The polynucleotidecan comprise a sequence of synthetic or naturally occurring DNA or RNAnucleotide bases.

As used herein, the term “selectable marker gene” refers to a gene,which upon its expression into a polypeptide in a cell, is detectabledue to a specific property of the polypeptide (e.g., enzymatic activityor fluorescence).

As used herein “an interfering RNA” refers to any double stranded orsingle stranded RNA sequence, capable—either directly or indirectly(i.e., upon conversion)—of inhibiting or down regulating gene expressionby mediating RNA interference. Interfering RNA includes but is notlimited to small interfering RNA (“siRNA”) and small hairpin RNA(“shRNA”). “RNA interference” refers to the selective degradation of asequence-compatible messenger RNA transcript.

As used herein “an shRNA” (small hairpin RNA) refers to an RNA moleculecomprising an antisense region, a loop portion and a sense region,wherein the sense region has complementary nucleotides that base pairwith the antisense region to form a duplex stem. Followingpost-transcriptional processing, the small hairpin RNA is converted intoa small interfering RNA by a cleavage event mediated by the enzymeDicer, which is a member of the RNase III family.

As used herein “an RNAi” (RNA interference) refers to apost-transcriptional silencing mechanism initiated by smalldouble-stranded RNA molecules that suppress expression of genes withsequence homology.

As used herein, the term “cell surface protein” refers to a protein thatis 5 present on the surface of a cell.

As used herein, the term “expansion” refers to the propagation of a cellor cells without terminal differentiation.

As used herein, the term “differentiation” refers to the developmentalprocess of lineage commitment. A “lineage” refers to a pathway ofcellular development, in which precursor or “progenitor” cells undergoprogressive physiological changes to become a specified cell type havinga characteristic function (e.g., nerve cell, muscle cell, or endothelialcell). Differentiation occurs in stages, whereby cells gradually becomemore specified until they reach full maturity, which is also referred toas “terminal differentiation.” A “terminally differentiated cell” is acell that has committed to a specific lineage, and has reached the endstage of differentiation (i.e., a cell that has fully matured).

As used herein, the term “isolated” refers to a stem cell or populationof daughter stem cells in a non-naturally occurring state outside of thebody (e.g., isolated from the body or a biological sample from thebody). The biological sample can include synovial fluid, blood (e.g.,peripheral blood), or tissue.

As used herein, the term “purified” as in a “purified cell” refers to acell that has been separated from the body of a subject but remains inthe presence of other cell types also obtained from the body of thesubject. By “substantially purified” is meant that the desired cells areenriched by at least 20%, more preferably by at least 50%, even morepreferably by at least 75%, and most preferably by at least 90% or even95%.

By a “population of cells” is meant a collection of at least ten cells.Preferably, the population consists of at least twenty cells, morepreferably at least one hundred cells, and most preferably at least onethousand, or even one million cells. Because the stem cells of thepresent invention exhibit a capacity for self-renewal, they can beexpanded in culture to produce populations of even billions of cells.

“Germ layers” are the three primary layers formed as a result ofgastrulation in early stage embryos, consisting of endoderm, mesoderm,and ectoderm. Embryonic germ layers are the source from which alltissues and organs derive. The endoderm is the source of, for example,pharynx, esophagus, stomach, intestine and associated glands (e.g.,salivary glands), liver, epithelial linings of respiratory passages andgastrointestinal tract, pancreas and lungs. The mesoderm is the sourceof, for example, smooth and

10 striated muscle, connective tissue, vessels, the cardiovascularsystem, blood cells, bone marrow, skeleton, reproductive organs andexcretory organs. Ectoderm is the source of, for example, epidermis(epidermal layer of the skin), sensory organs, the entire nervoussystem, including brain, spinal cord, and all the outlying components ofthe nervous system.

The term “multipotent,” with respect to stem cells of the invention,refers to the ability of the stem cells to give rise to cells of allthree primitive germ layers (endoderm, mesoderm, and ectoderm) upondifferentiation.

The term “allogeneic,” as used herein, refers to cells of the samespecies that differ genetically to the cell in comparison.

The term “autologous,” as used herein, refers to cells derived from thesame subject.

The term “engraft” as used herein refers to the process of stem cellincorporation into a tissue of interest in vivo through contact withexisting cells of the tissue.

By “does not express” means that expression of a protein or gene cannotbe detected by standard methods. In the case of cell surface markers,expression can be measured by flow cytometry, using a cut-off values asobtained from negative controls (i.e., cells known to lack the antigenof interest) or by isotype controls (i.e., measuring non-specificbinding of the antibody to the cell). Thus, a cell that “does notexpress” a marker appears similar to the negative control for thatmarker. For gene expression, a gene “does not express” if the presenceof its mRNA cannot be visually detected on a standard agarose gelfollowing standard PCR protocols.

Conversely, a cell “expresses” the protein or gene if it can be detectedby the same method.

As used herein, the term “loxP sites” refers to the consensus sitesrecognized by an enzyme of a bacteriophage (Cre-recombinase) inmediating site-specific recombination and excision.

As used herein, a “vector” or “expression vector” is a nucleicacid-based delivery vehicle comprising regulatory sequences and a geneof interest, which 10 can be used to transfer its contents into a cell.

A “subject” is a vertebrate, preferably a mammal (e.g., a non-humanmammal), more preferably a primate and still more preferably a human.Mammals include, but are not limited to, primates, humans, farm animals,sport animals, and pets.

By “embryoid body” is meant an aggregate of stem cells of the invention.In addition to expression of one or more of the transcription factorsdescribed herein (e.g., Oct-4, Nanog, Sox-2, Rex-1, GDF-3, and Stella),the cells in the embryoid body can also express KLF-4 or Myc.

The term “obtaining” as in “obtaining the stem cell” is intended toinclude purchasing, synthesizing or otherwise acquiring the stem cell(or indicated substance or material).

In this disclosure, the terms “comprises,” “comprising,” “containing”and “having” and the like can have the meaning ascribed to them in U.S.Patent law and can mean “includes,” “including,” and the like;“consisting essentially of or “consists essentially” likewise has themeaning ascribed in U.S. Patent law and the term is open-ended, allowingfor the presence of more than that which is recited so long as basic ornovel characteristics of that which is recited is not changed by thepresence of more than that which is recited, but excludes prior artembodiments.

Other definitions appear in context throughout the specification.

Stem Cells of the Invention

We have identified a quiescent adult stem cell having embryonic stemcell characteristics with the capacity to differentiate into ectoderm,mesoderm and endoderm, and having low immunogenic potential, as it doesnot express cell surface markers including MEC class I, MHC class II,CD44, CD45, CD13, CD34, CD49c, CD73, CD66b, CD105 and CD90.

The quiescent stem cell is in the resting phase of the cell cycle, Gap0(G0), and therefore, may not exhibit proliferative characteristics, suchas expression of the transcription factor Oct-4. This stem cell can befound in bodily fluids such as blood and synovial fluid or tissue. Theblood may be mobilized or non-mobilized blood.

Upon activation, the quiescent stem cell becomes proliferative, andexpresses genes including Oct-4 (Lau et al., Adv. Anat. Pathol.13:76-79, 2006), Nanog (Pan et al., J. Biol. Chem. 280:1401-1407, 2005),Sox2 (Lee et al., Cell 125:301-313, 2006), GDF3 (Hexige et al.,Neurosci. Lett. 389: 83-87, 2005), P 16INK4 (Gray-Schopfer et al., Br.J. Cancer; 95:496-505, 2006), BMI (Itahana, K., Mol. Cell. Biol.23:389-401, 2003), Notch (Chiang et al., Mol. Cell. Biol.; 26:6261-6271,2006), HDAC4 (Zeremski et al., Genesis 35: 31-38, 2003), TERT (Middlemanet al., Mol. Cell. Biol. 26:2146-2159, 2006), Rex-1 (Zhang et al., StemCells; 24:2669-2676, 2006), and TWIST (Guenou et al., Hum. Mol. Genet.14:1429-1439, 2005), but retains its low immunogenic potential, as itdoes not express cell surface markers including MEC class I, MEC classII, CD44, CD45, CD13, CD34, CD49c, CD66b, CD73, CD105 and CD90. The sizeof the cell has been observed to be about 6 gm to about 20 gm.

The proliferative stem cells of the invention are uniquely suited forlarge scale use. They are proliferative after less than ten days inculture, e.g., about one day, three days, or seven days in culture, anddo not require expansion in order to achieve an activated and/orproliferative state. Accordingly, it is possible to obtain about 5% toabout 30% proliferative stem cells from a population of about500,000-12,000,000 mobilized cells without expansion in vitro.

Stem cells of the invention are multipotent, having the capacity todifferentiate into a cell lineage of each germ layer (e.g., ectoderm,mesoderm, and endoderm). Upon further differentiation, the stem cellscan be fully differentiated into cell types including but not limited toa neuron, chondroblast, osteoblast, adipocyte, hepatocyte, smooth musclecell, skeletal muscle cell, cardiac cell, pancreatic cell, pulmonarycell, and endothelial cell. Methods for the differentiation of stemcells are well known in the art. Typically, stem cells are cultured inthe presence of differentiation-specific agents, which promote lineagecommitment. Differentiation-specific agents and conditions include, forexample, PDGF (e.g., about 10 ng/ml) and TGF-β1 (e.g., about 5 ng/ml)for the formation of a muscle cell; bFGF (e.g., about 100 ng/ml), FGF-8(e.g., about 10 ng/mL), SHH (e.g., about 100 ng/ml) and BDNF (e.g.,about 10 ng/ml) for the formation of a neuron; hepatocyte growth factor(HGF) and FGF-4 for the formation of a hepatocyte; dexamethasone,3-isobutyl-1-methylxanthine (IBMX), insulin and indomethacin for theformation of an adipocyte; VEGF (e.g., about 100 ng/ml of VEGF-165) forthe formation of an endothelial cell; and bone morphogenic protein-4(BMP4) (e.g., about 10 ng/ml), VEGF, bFGF, stem cell factor (SCF),Flt3L, hyper [L6, thrombopoietin (TPO), and erythropoietin (EPO) for theformation of a hematopoietic cell.

The stem cells of the invention can also form embryoid bodies uponculture. The cells forming the embryoid bodies, in addition to theembryonic transcription factors described herein, may additionallyexpress KLF-4 or Myc. Exemplary culture conditions for embryoid bodyformation are described in Example 4 below.

Isolation of Stem Cells

Any bodily source where stem cells of the invention are suspected ofresiding may be used for purification according to the methods describedherein. Methods of obtaining stem cells of the invention, particularlycells in 30 blood and synovial fluid, can be conducted as describedbelow.

Mobilization

Prior to removal of the cells from the subject, stem cells mayoptionally be mobilized using any method known in the art. Typically,stem mobilization is induced by administering an appropriate agent, suchas a cytokine or chemotherapeutic agent, to the subject. Cytokines thatcan be used to mobilize stem cells include G-CSF, GM-CSF, Flt-3 ligand,stem cell factor (SCF), IL-3 receptor agonists (e.g., Daniplestim),thrombopoietin agonists, chimeric cytokins (e.g., leridistim andprogenipoietin-1), peg-fligrastim, and SDF-1 antagonists (e.g., AMD3100). Chemotherapeutic agents include cyclophosphamide (Cy), orcombined chemotherapy regimens such as iphosphamide, carboplatin andetoposide (ICE) and etoposide, methylprednisolone, ara-c and cisplatin(ESHAP) (Cottler-Fox et al., Hematology Am Soc Hematol Educ Program pp.419-37, 2003).

Purification of Stem Cells

The stem cells of the invention can be purified from any bodily fluid ortissue in which they are found, including synovial fluid and blood. Insome embodiments, the stem cells of the invention are purified from thesynovial fluid of a subject suffering from osteoarthritis.

One bodily reservoir in which the stem cells reside is synovial fluid.To obtain the stem cells from the synovial fluid, cells can be spundown, pelleted, resuspended. The cells pelleted from synovial fluidcontains the stem cells of the invention. The cells from the pellet maybe cultured or may be further purified, e.g., by cell depletion or usinga discontinuous density gradient (e.g., DM-M, sucrose, or percollgradients). In one exemplary protocol, synovial fluid fromosteoarthritic patients is harvested, diluted in serum-free medium(AIM-V, GIBCO), and spun at about 200 g for about 15 minutes at roomtemperature. The pelleted population is then resuspended in AIM-V up tothe original synovial fluid volume. The cells are then counted with ahemacytometer. The resuspended, washed sample is layered over adiscontinuous density gradient (DM-M, Stem Cell Technologies Inc.) andspun at 500 g for about 30 minutes at room temperature. Thediscontinuous density gradient separates synovial fluid into a buffylayer and a pelleted layer. This gradient advantageously preventsgranulocytes from pelleting with the smaller cells (e.g., about 6 μm indiameter or less). Use of this gradient allows only two populations ofcells (RBC and stem cells) to form the pellet. The buffy layer is foundat the AIMV and DM-M interface while the pellet population is found inthe conical portion of the tube. The desired cellular population isisolated from the pellet layer, washed in PBS, and prepared for flowcytometry analysis, if desired.

Stem cells of the invention may also be isolated from blood (i.e.,hematopoietic tissue). Possible sources of human hematopoietic tissueinclude, but are not limited to, embryonic hematopoietic tissue, fetalhematopoietic tissue, and post-natal hematopoietic tissue. Embryonichematopoietic tissue can be yolk sac or embryonic liver. Fetalhematopoietic can be selected from fetal liver, fetal bone marrow andfetal peripheral blood. The post-natal hematopoietic can be cord blood,bone marrow, normal peripheral blood, mobilized peripheral blood,hepatic hematopoietic tissue, or splenic hematopoietic tissue.

In one exemplary protocol, blood from mammals is harvested, diluted inserum-free medium (AIM-V, GIBCO), and spun at about 200 g for 10 minutesat room temperature, about three times. The pelleted red cell fractioncan either be cultured or further enriched as described below. Furtherpurification may include resuspending the cells in AIM-V up to two orthree times the original volume. The resuspended, washed sample islayered over a combined gradient of Ficoll-Paque and Stem CellsTechnologies Granulocyte gradient (ROSETTE SEP DM-M, Stem CellTechnologies). This gradient advantageously prevents granulocytes frompelleting with the smaller cells (e.g., 6 μm in diameter), thus allowingonly two populations of cells (RBC and stem cells) to pellet. The sampleis subsequently spun at about 500 g for 30 minutes at room temperature.The Ficoll-Stem Cell Technology gradients separate the cells into abuffy layer, an intermediate layer, and pelleted layer. The desiredcellular population is isolated from the pelleted layer, washed in PBS,and resuspended in AIM V for further enrichment, if desired.

Further enrichment of the desired stem cells may be achieve by depletionof T cells (CD2/CD3), B cells (CD19/CD20), NK cells (CD16/CD56),dendritic cells (CD13,CD14, CD11B, WIC Class II), monocytes (CD13,CD14,CD11B Class II), granulocytes (CD13,CD14, CD66B), red blood cells, orany combination thereof.

Further separation can also be achieved using an anti-glycophorin Aantibody which binds most, if not all, red blood cells. This antibody isadded, and the red blood cells are depleted from the stem cellpopulation. Sorting can then be performed either by flow cytometric orimmunomagnetic bead selection. A variation of this method involves thedepletion of all cells except the stem cells by using the tetramericantibody complex, as described in U.S. Pat. No. 6,448,075.

Alternatively, red blood cells can be removed by CD71 and/orHoechst33258 (H0258) staining, followed by sorting using flow cytometry.Such a method is described in Tao et al., Zhonghua Yi Xue Yi Chuan XueZa Zhi. 17:352-354, 2000.

Another approach involves lysis of the red blood cells, and sortingusing electric fields, as described in U.S. Pat. No. 6,043,066.

Because the stem cells of the invention, when cultured with dendriticcells typically proliferate more rapidly, and such dendritic cells occurnaturally in synovial fluid, it may be desirable to purify the stemcells of the invention along with naturally occurring dendritic cells,e.g., using techniques which do not deplete dendritic cells from thestem cells of the invention.

Enrichment

Once a population of cells containing the desired stem cells has beenisolated from a subject, (e.g., according to the methods describedabove), 30 proliferating stem cells can be enriched. The proceduresdescribed above produce a mixed population of both quiescent andproliferating stem cells (FIGS. 1a-1e ). If desired, proliferative stemcells of the invention, which are proliferative after less than ten daysin culture and do not require expansion in order to achieve an activatedand/or proliferative state, can be enriched. The enrichment may beperformed using dyes (e.g., either negative or positive identificationof stem cells using such dyes such as those described herein), sortingtechniques, or by using a vector with a stem-cell specific promoteroperably linked to a detectable marker gene).

To perform the enrichment, a vector having a stem cell-specific promotercoupled to at least one selectable marker gene can be introduced into acell population (e.g., a population of mobilized stem cells obtainedfrom a source such as synovium or blood). Within the population are theproliferative stem cells that express Oct-4, Nanog, Sox 2, Rex-1, GDF-3and Stella, and do not express WIC class I, WIC class II, CD44, CD45,CD13, CD34, CD49c, CD73, CD105 and CD90 cell surface markers. Uponintroduction into the proliferative stem cell, the selectable markergene is expressed by internal factors that activate the stemcell-specific promoter. Detection of marker gene expression facilitatessorting and isolation of the cells according to methods known in the art(e.g., fluorescence activated flow cytometry and affinity beadpurification).

The stem cell-specific promoter can be any promoter that is capable ofdriving transcription of a gene in a multipotent stem cell, but not in alineage-committed or differentiated cell. Specificity for promoteractivation in a multipotent cellular environment allows for activationof selectable marker gene expression within the target proliferativestem cells, but not within more committed progenitors. Preferably, thestem cell-specific promoter is an Oct-4 promoter (Sylvester et al.,Nucleic Acids Res. 22:901-911, 1994), Nanog promoter (Wu da et al., CellRes. 15:317-324, 2005), Sox2 promoter (Boer, et al., Nucleic Acids Res.,35:1773-1786, 2007), Rex-1 promoter (Shi et al., J. Biol. Chem.,281:23319-23325, 2006), GDF-3 promoter (Clark, A. T., Stem Cells, 2004;22(2):169-79), Stella promoter (Clark, A. T., Stem Cells, 2004;22(2):169-79), FoxD3 promoter (Alkhateeb, A., J. Invest. Dermatol., 2005125, 388-391), Polycomb Repressor Complex 2 promoter (Sparmann, A., Nat.Rev., 2006. 6, 846-856, and CTCF promoter (De La Rosa-Velazquez I A,Cancer Res., 2007; 67(6):2577-85).

The selectable marker gene may be any known in the art, and ispreferably a gene which does not disrupt the growth and proliferation ofa live cell, such as a fluorescent protein, preferably a GreenFluorescent Protein (GFP). Where additional selectivity is desirable forisolation, the vector may encode multiple selectable marker genescoupled to one or more stem cell specific promoters. For example, thevector may encode a fluorescent protein and a protein sensitive to drugselection. The selectable marker gene may also encode a cell surfaceprotein which may be sorted according to standard methods known in theart.

The cells expressing the selectable marker gene from the stem-cellspecific promoter may be separated from each other by, for example,fluorescence activated cell sorting as disclosed herein. A preferredsorting procedure is by fluorescent activated cell sorting (FACS),wherein the cells can be separated on the basis of the level of stainingof the particular antigens. These techniques are well known to thoseskilled in the art and are described in various references includingU.S. Pat. Nos. 5,061,620; 5,409,8213; 5,677,136; and 5,750,397; and Yauet al., Exp. Hematol. 18:219-222 (1990).

In specific embodiments, the stem cell-specific promoter is flanked byloxP sites, so that the promoter may be conveniently excised to preventexpression of the selectable marker gene following isolation. It may beadvantageous to stop expression of the marker gene, for example, whereclinical use of the cells is desired.

Vectors for use in enrichment methods of the invention can be any knownin the art. Vectors containing both a promoter and a cloning site intowhich a heterologous polynucleotide can be operatively linked are wellknown in the art. One skilled in the art will readily recognize that anyheterologous polynucleotide can be excised as a compatible restrictionfragment and placed in a vector in such a manner as to allow properexpression of the heterologous polynucleotide from the stemcell-specific promoter. Such vectors are capable of transcribing RNA invitro or in vivo, and are commercially available from sources such asStratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.).Examples of vectors include vectors derived from viruses, such asbaculovirus, retroviruses, adenoviruses, adeno-associated viruses, andherpes simplex viruses; bacteriophages; cosmids; plasmid vectors; fungalvectors; synthetic vectors; and other recombination vehicles typicallyused in the art. These vectors have been used for expression in avariety of eukaryotic and prokaryotic hosts and may be used for proteinexpression. Specific non-limiting examples include pSG, pSV2CAT, andpXt1 from Stratagene (La Jolla, Calif.) and pMSG, pSVL, pBPV and pSVK3from Pharamacia. Other exemplary vectors include the pCMV mammalianexpression vectors, such as pCMV6b and pCMV6c (Chiron Corporation,Calif.), pSFFV-Neo, and pBluescript-SK+.

In order to optimize expression and/or in vitro transcription, it may benecessary to remove, add or alter 5′ and/or 3′ untranslated portions ofpolynucleotides to eliminate potentially extra inappropriate alternativetranslation initiation codons or other sequences that may interfere withor reduce expression, either at the level of transcription ortranslation. Alternatively consensus ribosome binding sites can beinserted immediately ‘5 of the start codon to enhance expression. Thevector may further comprise a polyadenylation signal that is positioned3′ of the carboxy-terminal amino acid.

The vector may be introduced into a cell using any method known in theart for introducing a nucleic acid into a cell and such methods arewell-known

25 in the art and are described, for example, in Sambrook et al. (1989,In: Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, New York), and Ausubel et al. (1997, In: CurrentProtocols in Molecular Biology, Green & Wiley, New York). These methodsinclude, but are not limited to, calcium phosphate precipitationtransfection, DEAE dextran transfection, electroporation,microinjection, liposome-mediated transfer, chemical-mediated transfer,ligand-mediated transfer, and recombinant viral vector transfer, and thelike.

In addition to or in place of cell enrichment using vector drivenexpression of various markers, additional cell enrichment techniques,including 5 depletion techniques and other sorting techniques, may beemployed.

One exemplary approach to enrich for the desired cells is magnetic beadcell sorting (MACS). The conventional MACS procedure is described byMiltenyi et al. (Cytometry 11:231-238, 1990). In this procedure, onelabels cells with magnetic beads and passes the cells through aparamagnetic separation column. The separation column is placed in astrong permanent magnet, thereby creating a magnetic field within thecolumn. Cells that are magnetically labeled are trapped in the column;cells that are not pass through. The trapped cells are then eluted fromthe column.

Stem cells of the invention can be enriched, for example, from asuitable bodily reservoir, such as the synovial fluid, using MACS toseparate MEC class I and glycophorin positive cells. The sample isincubated with immunomagnetic beads that bind to MHC class I and/orglycophorin.

Following incubation, samples are washed and resuspended at 10⁵-10⁶cells/ml and passed through a magnetic field to remove cells bound tothe immunomagnetic beads. Such negative selection techniques are knownto those of skill in the art. Monoclonal and polyclonal antibodiessuitable for negative selection purposes are also known to those ofskill in the art (see, for example, Leukocyte Typing V, Schlossman, etal., Eds. (1995) Oxford University Press), and are commerciallyavailable from a number of sources.

Other separation techniques, including affinity column chromatographyusing similar immunological reagents and FACS may also be used to removeparticular cell populations in order to enrich for the desired cells.

Another exemplary enrichment method involves incubating purified cellswith a detectable label that allow for sorting of cells. One exemplarylabel 30 is carboxyfluorescein diacetate, succinimidyl ester (CF SE).CFSE passively diffuses into cells. CFSE remains colorless andnonfluorescent until the acetate group is cleaved by intracellularesterases to yield high fluorescent carboxyfluorescein succinimidylester. The dye-protein adducts that form in labeled cells are retainedby the cells throughout development and meiosis, and can be used for invivo tracing. The label is inherited by daughter cells after either celldivision or cell fusion, and is not transferred to adjacent cells in apopulation. Because CFSE is partitioned equally among daughter cellswith each division, thus allowing simultaneous analysis of cell number,position, as well as division status. Based on this partitioning,proliferating stem cells can therefore be identified and separated basedon CFSE content.

An additional enrichment method involves the use of Aldefluor, afluorescent substrate for aldehyde dehydrogenase (ALDH). Human stem andprogenitor cells express high levels of ALDH activity when measured byflow cytometry, whereas differentiated cells do not. Primitivehematopoietic cells are relatively resistant to alkylating agents suchas the active derivatives of cyclophosphamide (e.g.,4-hydroxyperoxycyclophosphamide (4-HC) and mafosphamide). Thisresistance is due to the selective expression in primitive hematopoieticcells of the enzyme aldehyde dehydrogenase (ALDH). FluorescentALDH-substrates can be used to identify, quantitate and isolatehematopoietic cells by flow cytometry. Commercially available ALDEFLUORreagent systems offers a non-immunological way to identify human stemcells and progenitors from bone marrow (BM), mobilized peripheral blood(MPB) and umbilical cord blood (UCB) on the basis of their ALDHactivity.

In various embodiments of the invention, multiple enrichment strategiescan be combined to achieve isolation of proliferative stem cells. Forexample, vector-based isolation techniques can be used together withimmunomagnetic sorting, either before or after vector-based sorting, andeither with or without subsequent FACS analysis. In one exemplaryprotocol, purified cells that are MHC class I and glycophorin negative,obtained by negative selection methods described herein above, areresuspended to 10⁵ per ml and placed in 6-well tissue culture plastic. Alentiviral vector expressing GFP under the control of an Oct-4 stemcell-specific promoter is added to the cells and cultured for one day,for three days, five days, and seven days. The GFP-expressing cells arethen subsequently sorted into individual wells by flow cytometry.

Culture

Stem cells of the invention can be maintained under standard cellculture conditions. For example, the cells can be maintained in DulbeccoMinimal Essential Medium (DMEM) or any other appropriate cell culturemedium, supplemented with 1-50 ng/ml (e.g., about 5-15 ng/ml)platelet-derived growth factbr-BB (PDGF-BB), 1-50 ng/ml (e.g., about5-15 ng/ml) epidermal growth factor (EGF), 1-50 ng/ml (e.g., about 5-15ng/ml) insulin-like growth factor (IGF), or 100-10,000 IU (e.g., about1,060) LW, with 10⁻¹⁰ to 10⁻⁸M dexamethasone or other appropriatesteroid, 2-10 μg/ml linoleic acid, and 0.05

0.15 μm ascorbic acid. Additional culture conditions can be identifiedby one of skill in the art.

In one example, about 50,000 cells are grown under suitable conditions.The cells can be plated in fibronectin-coated wells of 96 well plates indefined medium consisting of 1% PHS, 10 ng/ml IGF, 10 ng/ml EGF and 10ng/ml PDGF-BB as well as transferrin, selenium, dexamethasone, linoleicacid, insulin, and ascorbic acid. The negatively-selected samples, whichcan optimally comprise a population of cells that is greater than 98%class I and glycophorin negative, can then be assessed for expression ofadult and embryonic stem cell markers, as well as the lack of expressionof MHC class I, MHC class II, CD44, CD45, CD13, CD34, CD49c, CD73,CD105, and CD90 cell surface markers, according to methods known in theart to confirm the identity of the purified population.

In specific embodiments, pooled human serum (1-2%) and human growthfactors are used to supplement growth and proliferation. Preferably,stem cells of the invention are grown in the presence of 1-2% pooledhuman serum, epidermal growth factor, and platelet-derived growthfactor-BB.

Other appropriate media include, for example, MCDB, Minimal EssentialMedium (MEM), IMDM, and RPML

Minimum Essential Medium (MEM) is one of the most widely used of allsynthetic cell culture media. Early attempts to cultivate normalmammalian fibroblasts and certain subtypes of HeLa cells revealed thatthey had specific nutritional requirements that could not be met byEagle's Basal Medium (BME). Subsequent studies using these and othercells in culture indicated that additions to BME could be made to aidgrowth of a wider variety of fastidious cells. MEM, which incorporatesthese modifications, includes higher concentrations of amino acids sothat the medium more closely approximates the protein composition ofmammalian cells. MEM has been used for cultivation of a wide variety ofcells grown in monolayers. Optional supplementation of non-essentialamino acids to the formulations that incorporate either Hanks' orEagles' salts has broadened the usefulness of this medium. Theformulation has been further modified by optional elimination of calciumto permit the growth of cells in suspension.

Iscove's Modified Dulbecco's Media (IMDM) is a highly enriched syntheticmedia. IMDM is well suited for rapidly proliferating, high-density cellcultures.

MCDB media were developed for the low-protein and serum free growth ofspecific cell types using hormones, growth factors, trace elementsand/or low levels of dialyzed fetal bovine serum protein (FBSP). EachMCDB medium was formulated (quantitatively and qualitatively) to providea defined and optimally balanced nutritional environment thatselectively promoted the growth of a specific cell line. MCDB 105 and110 are modifications of MCDB 104 medium, optimized for long-termsurvival and rapid clonal growth of human diploid fibroblast-like cells(WI-38, MRC-5, IMR-90) and low passaged human foreskin fibroblasts usingFBSP, hormone, and growth factor supplements. MCDB 151, 201, and 302 aremodifications of Ham's nutrient mixture F-12, designed for the growth ofhuman keratinocytes, clonal growth of chicken embryo fibroblasts (CEF)and Chinese hamster ovary (CHO) cells using low levels of FBSP,extensive trace elements or no serum protein.

RPMI-1640 was developed by Moore et. al. at Roswell Park MemorialInstitute, hence the acronym RPMI. The formulation is based on theRPMI-1630 series of media utilizing a bicarbonate buffering system andalterations in the amounts of amino acids and vitamins. RPMI-1640 mediumhas been used for the culture of human normal and neoplastic leukocytes.RPMI-1640, when properly supplemented, has demonstrated wideapplicability for supporting growth of many types of cultured cells,including fresh human lymphocytes in the 72 hour phytohemaglutinin (PHA)stimulation assay.

Stem cells of the invention can be maintained according to culturemethods known in the art enhance proliferation. Preferably,proliferative stem cells of the invention are plated infibronectin-coated wells of 96 well plates in defined medium consistingof 1% PHS, 10 ng/ml IGF, 10 ng/ml EGF and 10 ng/ml PDGF-BB as well astransferrin, selenium, dexamethasone, linoleic acid, insulin, andascorbic acid.

To improve proliferation of these cells, the stem cells of the inventioncan be co-cultured with dendritic cells or antigen-presenting cells.These co-cultures can be carried out using basal or propagation cultureconditions, as described herein. Dendritic cells can also be culturedusing 10% pooled human serum (PHS) in standard culture medium plusantibiotics. We have observed that use of human serum results in thestem cells growing better (e.g., in 1%-2% PHS) as compared to bovineserum. Alternatively, serum free media may be used.

In other embodiments, the cells may be cultured in the presence of anextracellular matrix. Suitable procedures for proliferating cells in thepresence 25 of such matrices are described, for example, in U.S. Pat.No. 7,297,539.

Methods of Use

The production of stem cells, which can be either maintained in anundifferentiated state or directed to undergo differentiation intoextraembryonic 30 or somatic lineages ex vivo, allows for the study ofthe cellular and molecular biology of events of early human development,generation of differentiated cells from the stem cells for use intransplantation (e.g., autologous or allogenic transplantation),treating diseases (e.g., any described herein), tissue generation,tissue engineering, in vitro drug screening or drug discovery, andcryopreservation.

Transplant and Treatment of Disease

The stem cells of the invention may be used in autologous or allogenicstem cell transplantation. Stem cell transplantation is a usefulapproach of repairing damaged tissue, for example, in the treatment ofdiseases including, but not limited to, cardiac disease,neurodegenerative disease, diabetes, wound healing, diseases treatableby immunosuppression (e.g., autoimmune disorders), and bone andcartilage replacement or augmentation.

Stem cells of the invention have the potential to differentiate into avariety of cell types including but not limited to a neuron,chondroblast, osteoblast, adipocyte, hepatocyte, muscle cell (e.g.,smooth muscle or skeletal muscle), cardiac cell, pancreatic cell,pulmonary cell, and endothelial cell. Accordingly, stem cells of theinvention can be transplanted into a subject, engrafted into a targettissue, and differentiated in vivo to match the tissue type andsupplement the target tissue, thereby restoring or enhancing function.In other cases, a stem cell is differentiated into a particular targettissue prior to transplantation.

Cardiac

The stem cells of the invention may be used in the treatment of cardiacconditions, e.g., where cardiac tissue has been damaged. Exemplaryconditions include myocardial infarction, congestive heart failure,ischemic cardiomyopathy, and coronary artery disease. Such methods aredescribed, for example, in U.S. Pat. No. 6,534,052, incorporated hereinby reference in its entirety. Here, embryonic cells are introducedsurgically and implanted into the infarcted area of the myocardium.After implantation, the embryonic stem cells form stable grafts andsurvive indefinitely within the infarcted area of the heart in theliving host. In other cases, the cells are cultured under conditionsthat induce differentiation into cardiac tissue prior totransplantation.

Vascularization

The stem cells of the invention may also be used to increasevascularization. Doing so may be desirable when organs have been injuredor in cases of diabetic disorders. Diseases in which increasedvascularization is desirable include diabetes, atherosclerosis,arteriosclerosis, and any of the cardiac conditions described above.

Neurological Conditions

Stem cells of the invention or their committed or differentiated progenymay also be used to treat neural disorders where regeneration of tissueis desirable. Stem cells of the invention can address the shortage ofdonor tissue for use in transplantation procedures, particularly whereno alternative culture system can support growth of the requiredcommitted stem cell. In another example, following transplantation intothe central nervous system (CNS), ES cell-derived neural precursors havebeen shown to integrate into the host tissue and, in some cases, yieldfunctional improvement (McDonald et al., Nat. Med. 5:1410-1412, 1999).

Neurological diseases that can be treated using stem cells of theinvention include neurodegenerative disorders such as Parkinson'sdisease, polyglutamine expansion disorders (e.g., Huntington's Disease,dentatorubropallidoluysian atrophy, Kennedy's disease (also referred toas spinobulbar muscular atrophy), and spinocerebellar ataxia (e.g., type1, type 2, type 3 (also referred to as Machado-Joseph disease), type 6,type 7, and type 17)), other trinucleotide repeat expansion disorders(e.g., fragile X syndrome, fragile XE mental retardation, Friedreich'sataxia, myotonic dystrophy, spinocerebellar ataxia type 8, andspinocerebellar ataxia type 12), Alexander disease, Alper's disease,Alzheimer's disease, amyotrophic lateral sclerosis, ataxiatelangiectasia, Batten disease (also referred to asSpielmeyer-Vogt-Sjogren-Batten disease), Canavan disease, Cockaynesyndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, ischemiastroke, Krabbe disease, Lewy body dementia, multiple sclerosis, multiplesystem atrophy, Pelizaeus-Merzbacher disease, Pick's disease, primarylateral sclerosis, Refsum's disease, Sandhoff disease, Schilder'sdisease, spinal cord injury, brain injury, spinal muscular atrophy,Steele-Richardson-Olszewski disease, and Tabes dorsalis.

Immunosuppression and Treatment of Autoimmune Diseases

Stem cells of the invention may also be used to inhibit or reduceundesired or inappropriate immune responses. For example, the stem cellsmay be used to treat an autoimmune disease, to promote wound healing, orto reduce or prevent rejection of a tissue or organ. Stem cells can beused to suppress immune responses upon administration to subjects. See,e.g., U.S. Patent Application Publication No. 2005/0282272. Suchapproaches have also been proposed in Sykes et al., Nature 435:620-627,2005 and Passweg et al., Semin. Hematol. 44:278-85, 2007. Otherimmunosuppressive uses of stem cells are described in U.S. Pat. Nos.6,328,960, 6,368,636, 6,685,936, 6,797,269, 6,875,430, and 7,029,666.

Thus, stem cells of the invention may be used for immunosuppression orto treat autoimmune disease. Immunosuppression may be desirable prior totransplantation of tissues or organs into a patient (e.g., thosedescribed herein). Autoimmune disease which may be treated byadministration of stem cells include multiple sclerosis (MS), systemicsclerosis (SSc), systemic lupus erythematosus (SLE), rheumatoidarthritis (RA), juvenile idiopathic arthritis, and immune cytopenias.Other autoimmune disease which may be treated using stem cells of theinclude acute disseminated encephalomyelitis (ADEM), Addison's disease,Ankylosing spondylitis, antiphospholipid antibody syndrome (APS),aplastic anemia, autoimmune hepatitis, autoimmune oophoritis, celiacdisease, Crohn's disease, diabetes mellitus type 1, gestationalpemphigoid, Goodpasture's syndrome, Graves' disease, Guillain-Barrésyndrome (GBS), Hashimoto's disease, idiopathic thrombocytopenicpurpura,

Kawasaki's disease, lupus erythematosus, mixed connective tissuedisease, multiple sclerosis, myasthenia gravis, opsoclonus myoclonussyndrome, Ord's thyroiditis, pemphigus, pernicious anaemia, primarybiliary cirrhosis, rheumatoid arthritis, Reiter's syndrome, Sjogren'ssyndrome, Takayasu's arteritis, temporal arteritis, warm autoimmunehemolytic anemia, and Wegener's granulomatosis.

In other embodiments, the stem cells of the invention can be used toreduce or prevent rejection of a transplanted tissue or organ. Forinstance, such a method can include engrafting the hematopoietic systemof the tissue or organ recipient with stem cells of the inventionobtained from the organ donor prior to transplanting the organ. Byengrafting the hematopoietic system of the recipient with stem cellsderived from the organ donor, rejection of the transplanted organ isthereby inhibited. Prior to engraftment and organ transplantation, thebone marrow of the recipient would be ablated by standard methods wellknown in the art. Generally, bone marrow ablation is accomplished byX-radiating the animal to be transplanted, administering drugs such ascyclophosphamide or by a combination of X-radiation and drugadministration. Bone marrow ablation can be produced by administrationof radioisotopes known to kill metastatic bone cells such as, forexample, radioactive strontium, ¹³⁵Samarium, or ¹⁶⁶Holmium (Applebaum etal., 1992, Blood 80:1608-1613).

In some embodiments, autologous stem cells can be transplanted into asubject. A population of stem cells can be isolated from the recipientaccording to the methods described herein prior to ablating bone marrowof the recipient.

The bone marrow of the individual is purged of malignant blasts andother malignant cells such that by transplanting the non-malignant stemcells back into to the individual, diseases such as melanomas may betreated.

Wound Healing

The stem cells of the invention can also be used to improve woundhealing. Inflammation during healing of wounds has been shown toincrease scarring at wound sites (Redd et al., Philos. Trans. R. Soc.Lond. B Biol. Sci. 359:777-784, 2004). Doing so at a wound site canpromote healing of the tissue and further can decrease fibrosis andscarring at the wound site. Because formation of age-related wrinklesmay also be caused by a scarring process, administration of stem cellsof the invention to the site of wrinkles may reduce wrinkle formation orresult in reduction or elimination of such of wrinkles as well as scars.Wound healing using regenerative cells from adipose tissue is described,for example, in U.S. Patent Application Publication Nos. 2005/0048034and 2006/0147430. Such approaches can be adapted for use with the cellsof the present invention.

Tissue Generation

Stem cells of the invention may also be used in promoting tissuegeneration, e.g., to replace damaged or diseased tissue. The term“promoting tissue generation” includes activating, enhancing,facilitating, increasing, inducing, initiating, or stimulating thegrowth and/or proliferation of tissue, as well as activating, enhancing,facilitating, increasing, inducing, initiating, or stimulating thedifferentiation, growth, and/or proliferation of tissue cells. Thus, theterm includes initiation of tissue generation, as well as facilitationor enhancement of tissue generation already in progress. The term“generation” also includes the generation of new tissue and theregeneration of tissue where tissue previously existed.

Stem cells of the invention are multipotent, and have the potential todifferentiate into a variety of cell types as discussed above. As such,the cells are useful in tissue generation. For instance, the stem cellsof the invention can be used in the generation of neural cells. Morespecifically, stem cells of the invention can be induced todifferentiate into neural cells using, for example, commerciallyavailable products such as NEUROCULT (Stem Cell Technologies).

Stem cells of the invention may be used in the production of tissuesaccording to methods known in the art. U.S. Pat. No. 5,834,312,incorporated by reference in its entirety herein, for example, describesmedia and methods for the in vitro formation of a histologicallycomplete human epithelium. The media are serum-free, companion cell orfeeder layer free and organotypic, matrix free solutions for theisolation and cultivation of clonally competent basal epithelial cells.The media and methods of the invention are useful in the production ofepithelial tissues such as epidermis, cornea, gingiva, and ureter. U.S.Pat. No. 5,912,175, incorporated by reference in its entirety herein,describes media and methods for the in vitro formation of human corneaand gingival from stem cells.

U.S. Pat. No. 6,497,872, incorporated by reference in its entiretyherein, describes the differentiation of stem cells into neural cells(e.g., neurons, astrocytes, and oligodendrocytes), and methods forneurotransplantation in the undifferentiated or differentiated state,into a subject to alleviate the symptoms of neurological disease,neurodegeneration and central nervous system (CNS) trauma. Methods forthe generation of suitable in autografts, xenografts, and allografts arealso described.

In certain embodiments, it may be desirable to treat the cells in orderto decrease the likelihood of transplant rejection, especially wherenon-autologous cells are used. The invention therefore features methodsof decreasing uric acid production in cells, and cells in which uricacid production has been reduced.

Exemplary means for doing so are described in U.S. Patent ApplicationPublication No. 2005/0142121 and include treatment with compounds thatdecrease xanthine oxidase activity, such as allopurinol, oxypurinol, andBOF-4272. Other approaches include pre-treatment with low levels oftungsten to deplete molybdenum, a necessary cofactor for xanthineoxidase. Genetic or RNAi approaches which reduce transcription ortranslation of the xanthine oxidase gene or mRNA, may also be used todecrease uric acid production.

Stem cells of the invention may also be used for the generation oftissue engineered constructs or grafts, such as for use in replacementof bodily tissues and organs (e.g., fat, liver, smooth muscle,osteoblasts, kidney, liver, heart, and neural tissue). Stem cells of theinvention may also be particularly well suited for the generation oftissue engineered constructs for use in replacement of musculoskeletaltissues (e.g., cartilage, joint, ligament, tendon).

For instance, the inability to use articular cartilage for self-repairis a major problem in the treatment of patients who have their jointsdamaged by traumatic injury or suffer from degenerative conditions, suchas arthritis or osteoarthritis. New approaches to cartilage tissuerepair based on implanting or injecting expanded autologous cells into apatient's injured cartilage tissue can be used. More recently, it hasbeen proposed in EP-A-0 469 070, incorporated by reference herein in itsentirety, to use a biocompatible synthetic polymeric matrix seeded withchondrocytes, fibroblasts or bone-precursor cells as an implant forcartilaginous structures. Stem cells of the invention can bedifferentiated into chondroblasts, and optionally seeded on a matrix forimplantation into a patient in need of cartilage replacement. A suitablematrix is described, for example, in U.S. Pat. No. 6,692,761,incorporated by reference in its entirety herein, in a material that hashydrogel properties and allows for diffusion through the materialitself, in addition to diffusion through its porous structure. Thisfeature is highly advantageous when cells are seeded onto the scaffoldand are cultured thereon, as it enables a very efficient transport ofnutrient and waste materials from and to the cells. Secondly, thematerial closely mimics the structure and properties of naturalcartilage, which, containing 80% water, is also a hydrogel. Other matrixcell based cultures are described in U.S. Pat. Nos. 5,855,619 and5,962,325.

Methods of transplanting stem cells, stem cell-derived progeny (e.g.,differentiated cells) and/or stem cell-derived tissue grafts are wellknown in the art. For example, U.S. Pat. No. 7,166,277, (“the '277patent”), incorporated by reference in its entirety herein, describesthe use of stem cells and their progeny as neuronal tissue grafts. Themethods taught in the '277 patent for the in vitro proliferation anddifferentiation of stem cells and stem cell progeny into neurons and/orglia for the treatment of neurodegenerative diseases can be applied tothe stem cells of the invention. Differentiation occurs by exposing thestem cells to a culture medium containing a growth factor which inducesthe cells to differentiate. Proliferation and/or differentiation can bedone before or after transplantation, and in various combinations of invitro or in vivo conditions, including (1) proliferation anddifferentiation in vitro, then transplantation, (2) proliferation invitro, transplantation, then further proliferation and differentiationin vivo, and (3) proliferation in vitro, transplantation anddifferentiation in vivo. As another example, U.S. Pat. No. 7,150,990,incorporated by reference in its entirety herein, describes methods fortransplanting stem cells and/or stem cell-derived hepatocytes into asubject to supplement or restore liver function in vivo. Such methodscan also be applied to the stem cells of the invention. As yet anotherexample, U.S. Pat. No. 7,166,464, incorporated by reference in itsentirety herein, provides methods for the formation of a tissue sheetcomprised of living cells and extracellular matrix formed by the cells,whereby the tissue sheet can be removed from the culture container togenerate a genetically engineered tissue graft. Practitioners can followstandard methodology known in the art to transform the stem cells of theinvention into a desired cell type or engineered construct for use intransplantation.

Stem cells of the invention may be used to produce muscle cells (e.g.,for use in the treatment of muscular dystrophy (e.g., as Duchenne's andBecker's muscular dystrophy and denervation atrophy). See, e.g., U.S.Patent 25 Application Publication No. 2003/0118565.

Gene Therapy

The stem cells of the invention may be transfected for use in genetherapy applications. Stem cells of the invention may be transfectedusing any 30 methods known in the art such as viral vector systems,microinjection, electroporation, liposomes, and chromosome transfer, orany other method described herein may be used.

A wide variety of nucleic acids may be transfected into the stem cellsof the invention. Thus, the invention should be construed to includenucleic acid products which are useful for the treatment of variousdisease states in a mammal. Such nucleic acids and associated diseasestates include, but are not limited to: DNA encodingglucose-6-phosphatase, associated with glycogen storage deficiency type1A; DNA encoding phosphoenolpyruvate-carboxykinase, associated withPepck deficiency; DNA encoding galactose-1 phosphate uridyl transferase,associated with galactosemia; DNA encoding phenylalanine hydroxylase,associated with phenylketonuria; DNA encoding branched chain a-ketoaciddehydrogenase, associated with Maple syrup urine disease; DNA encodingfumarylacetoacetate hydrolase, associated with tyrosinemia type 1; DNAencoding methylmalonyl-CoA mutase, associated with methylmalonicacidemia; DNA encoding medium chain acyl CoA dehydrogenase, associatedwith medium chain acetyl CoA deficiency; DNA encoding omithinetranscarbamylase, associated with omithine transcarbamylase deficiency;DNA encoding argininosuccinic acid synthetase, associated withcitrullinemia; DNA encoding low density lipoprotein receptor protein,associated with familial hypercholesterolemia; DNA encodingUDP-glucouronosyltransferase, associated with Crigler-Najjar disease;DNA encoding adenosine deaminase, associated with severe combinedimmunodeficiency disease; DNA encoding hypoxanthine guaninephosphoribosyl transferase, associated with Gout and Lesch-Nyansyndrome; DNA encoding biotinidase, associated with biotinidasedeficiency; DNA encoding beta-glucocerebrosidase, associated withGaucher disease; DNA encoding beta-glucuronidase, associated with Slysyndrome; DNA encoding peroxisome membrane protein 70 kDa, associatedwith Zellweger syndrome; DNA encoding porphobilinogen deaminase,associated with acute intermittent porphyria; DNA encoding antitrypsinfor treatment of alpha-1 antitrypsin deficiency (emphysema); DNAencoding erythropoietin for treatment of anemia due to thalassemia or torenal failure; and, DNA encoding insulin for treatment of diabetes. SuchDNAs and their associated diseases are reviewed in Kay et al. (1994,T.I.G. 10:253-257) and in Parker and Ponder (1996, “Gene Therapy forBlood Protein Deficiencies,” In: Gene Transfer in CardiovascularBiology: Experimental Approaches and Therapeutic Implications, Keith andMarch, eds.).

Where a vector includes coding sequences in addition to those encodingselectable marker genes, such additional coding sequences may beoperably linked to a separate promoter/regulatory sequence. Manypromoter/regulatory sequences useful for driving constitutive expressionof a gene are available in the art and include, but are not limited to,for example, the cytomegalovirus immediate early promoter enhancersequence, the SV40 early promoter, the Rous sarcoma virus promoter, andthe like. Inducible and tissue specific expression of the nucleic acidoperably linked thereto may be accomplished by placing the nucleic acidunder the control of an inducible or tissue specific promoter/regulatorysequence. Examples of tissue specific or inducible promoter/regulatorysequences which are useful for this purpose include, but are not limitedto the MMTV long terminal repeat (LTR) inducible promoter, and the SV40late enhancer/promoter. In addition, promoters which are well known inthe art which are induced in response to inducing agents such as metals,glucocorticoids, and the like, are also contemplated in the invention.Thus, it will be appreciated that the invention should be construed toinclude the use of any promoter/regulator sequence which is either knowor is heretofore unknown, which is capable of driving expression of thenucleic acid operably linked thereto.

One skilled in the art will appreciate, based upon the disclosureprovided herein, that stem cells of the invention are useful for celltherapy. That is, such a stem cell would, when introduced into ananimal, express the nucleic acid thereby providing a method of producinga protein (or disrupting expression of an undesired protein through theuse of an interfering RNA), thus correcting a genetic defect in a cell,encode a protein which is not otherwise present in sufficient and/orfunctional quantity such that it corrects a genetic defect in the cell,and/or encodes a protein which is useful as a therapeutic in thetreatment or prevention of a particular disease condition or disorder orsymptoms associated therewith. Thus, stem cells of the invention areuseful therapeutics allowing the expression of an isolated nucleic acidpresent in such cell.

Stem cells of the invention can be genetically modified to express oneor more RNA interference (RNAi) molecules when administered to a patient(e.g., a human). RNAi is a mechanism that inhibits gene expression bycausing the degradation of specific RNA molecules or hindering thetranscription of specific genes. Key to the mechanism of RNAi are smallinterfering RNA strands (siRNA), which have complementary nucleotidesequences to a targeted messenger RNA (mRNA) molecule. siRNAs are short,single-stranded nucleic acid molecule capable of inhibiting ordown-regulating gene expression in a sequence-specific manner; see, forexample, Zamore et al., Cell 101:25 33 (2000); Bass, Nature 411:428-429(2001); Elbashir et al., Nature 411:494-498 (2001); and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914. Methods of preparing a siRNA molecule foruse in gene silencing are described in U.S. Pat. No. 7,078,196, which ishereby incorporated by reference.

The application of RNAi technology (e.g., an siRNA molecule) in thepresent invention can occur in several ways, each resulting infunctional silencing of a gene product in a stem cell population. Thefunctional silencing of one or more endogenous stem cell gene productsmay increase the longevity the stem cell in vivo (e.g., by silencing oneor more pro-apoptotic gene products), or increase the expression of atherapeutic polypeptide (e.g., an antibody, cytokine, or hormone).

Functional gene silencing by an RNAi agent (e.g., an siRNA molecule)does not necessarily include complete inhibition of the targeted geneproduct. In some cases, marginal decreases in gene product expressioncaused by an RNAi agent can translate to significant functional orphenotypic changes in the host cell, tissue, organ, or animal.Therefore, gene silencing is understood to be a functional equivalentand the degree of gene product degradation to achieve silencing maydiffer between gene targets or host cell type. Gene silencing maydecrease gene product expression by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,or 10%. Preferentially, gene product expression is decreased by 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% (i.e., completeinhibition).

Recombinant expression of non-endogenous polypeptides oroligonucleotides in stem cells of the invention can be accomplished byusing any standard gene transfer technique, examples of which arediscussed below.

In some embodiments, viral transduction can be used to geneticallymodify a stem cell of the invention. Many viruses bind and infectmammalian cells and introduce their genetic material into the host cellas part of their replication cycle. Some types of viruses (e.g.,retroviruses) integrate their viral genomes into the host's genome. Thisincorporates the genes of that virus among the genes of the host cellfor the life span of that cell. In viruses modified for gene transfer, adonor gene (e.g., a humanized monoclonal antibody) is inserted into theviral genome. Additional modifications are made to the virus to improveinfectivity or tropism (e.g., pseudotyping), reduce or eliminatereplicative competency, and reduce immunogencity. The newly-introducedmammalian gene will be expressed in the infected host cell or organismand, if replacing a defective host gene, can ameliorate conditions ordiseases caused by the defective gene. Adenoviruses and retroviruses(including lentiviruses) are particularly attractive modalities for genetherapy applications, as discussed below, due to the ability togenetically-modify and exploit the life cycle of these viruses.

In some embodiments, an adenoviral vector is used. Recombinantadenoviral vectors offer several significant advantages for theexpression of polypeptides (e.g., an antibodies, cytokines, or clottingfactors) or oligonucleotides (e.g., an siRNA) in stem cells of theinvention. The viruses can be prepared at extremely high titer, infectnon-replicating cells, and confer high-efficiency and high-leveltransduction of target cells in vivo after directed injection orperfusion. Furthermore, as adenoviruses do not integrate their DNA intothe host genome, there is a reduced risk of inducing spontaneousproliferative disorders. In animal models, adenoviral gene transfer hasgenerally been found to mediate high-level expression for approximatelyone week. The duration of transgene expression may be prolonged, andectopic expression reduced, by using tissue-specific promoters. Otherimprovements in the molecular engineering of the adenoviral vectoritself have produced more sustained transgene expression and lessinflammation. This is seen with so-called “second generation” vectorsharboring specific mutations in additional early adenoviral genes and“gutless” vectors in which virtually all the viral genes are deletedutilizing a cre-lox strategy (Engelhardt et al., Proc. Natl. Acad. Sci.USA 91:6196-6200 (1994) and Kochanek et al., Proc. Natl. Acad. Sci. USA93:5731-5736 (1996)). In addition, recombinant adeno-associated viruses(rAAV), derived from non-pathogenic parvoviruses, can be used to expressa polypeptide or oligonucleotide, as these vectors evoke almost nocellular immune response, and produce transgene expression lastingmonths in most systems. Incorporation of a tissue-specific promoter mayalso be beneficial.

Other viral vectors useful for the delivery of polypeptides oroligonucleotides into a subject or cells are retroviruses, includinglentiviruses. As opposed to adenoviruses, the genetic material inretroviruses is RNA, while the genetic material of their hosts is in theform of DNA. When a retrovirus infects a host cell, it introduces itsRNA together with enzymes into the cell. This RNA molecule is used toproduce a double-stranded DNA copy (provirus) by reverse transcription.Following transport into the cell nucleus, the proviral DNA isintegrated in a host chromosome, permanently altering the genome of theinfected cell and any progeny cells that may arise. Retroviruses includelentiviruses, a family of viruses including human immunodeficiency virus(HIV) that includes several accessory proteins to facilitate viralinfection and proviral integration.

One problem with using retroviruses for gene therapy is that theintegrase enzyme can insert the genetic material of the virus in anarbitrary position in the host genome, risking gene disruption (e.g.,insertional mutagenesis). If the gene happens to be one regulating celldivision, uncontrolled cell division (e.g., cancer) can occur. Toaddress this problem, inclusion of zinc finger nucleases or certainsequences, such as the beta-globin locus control region, are used todirect the site of integration to specific chromosomal sites. Current,“third-generation” lentiviral vectors feature total replicationincompetence, broad tropism, and increased gene transfer capacity formammalian cells (see Mangeat et al., Human Gene Therapy 16:913-920(2005) and Wiznerowicz et al., Trends Biotechnol. 23:42-7 (2005)).Lentiviruses pseudotyped with, e.g., vesicular stomatitis virusglycoprotein (VSV-G) or feline endogenous virus RD114 envelopeglycoprotein can be used to transduce stem cells of the invention. (see,e.g., Zhang et al., J. Virol. 78:1219-1229 (2004)). U.S. Pat. Nos.5,919,458, 5,994,136, and 7,198,950, hereby incorporated by reference,describe the production and use of lentiviruses to genetically modifytarget cells.

Besides adenoviral and retroviral vectors, other viral vectors andtechniques are known in the art that can be used to transfer a DNAvector (e.g., a plasmid) encoding a desired polypeptide oroligonucleotide into a subject or cells. These include, e.g., thosedescribed by Wattanapitayakul and Bauer (Biomed. Pharmacother 54:487-504(2000), and citations therein.

Other transfections approaches, including naked DNA or oligonucleotides(e.g., DNA vectors such as plasmids) encoding polypeptides (e.g., anantibody, cytokine, or hormone) or RNA interference molecule (e.g., ansiRNA or shRNA), can be used to genetically modify stem cells of theinvention. Improved transfection efficiency of nake DNA can be achievedusing electroporation or a “gene gun,” which shoots DNA-coated goldparticles into the cell using high pressure gas.

To improve the delivery of a DNA vector (e.g., a plasmid) into a stemcell of the invention, the DNA can be protected from damage and itsentry into the cell facilitated, for example, by using lipoplexes andpolyplexes. Plasmid DNA can be covered with lipids in an organizedstructure like a micelle or a liposome. When the organized structure iscomplexed with DNA it is called a lipoplex. There are three types oflipids, anionic (negatively-charged), neutral, or cationic(positively-charged). Lipoplexes that utilize cationic lipids haveproven utility for gene transfer. Cationic lipids, due to their positivecharge, naturally complex with the negatively charged DNA. Also as aresult of their charge, they interact with the cell membrane.Endocytosis of the lipoplex then occurs, and the DNA is released intothe cytoplasm. The cationic lipids also protect against degradation ofthe DNA by the cell.

Complexes of polymers with DNA are called polyplexes. Most polyplexesconsist of cationic polymers and their production is regulated by ionicinteractions. One large difference between the methods of action ofpolyplexes and lipoplexes is that polyplexes cannot release their DNAload into the cytoplasm, so to this end, co-transfection withendosome-lytic agents (to lyse the endosome that is made duringendocytosis) such as inactivated adenovirus must occur. However, this isnot always the case; polymers such as polyethylenimine have their ownmethod of endosome disruption as does chitosan and trimethylchitosan.

Hybrid methods have been developed that combine two or more techniquesdescribed above. Virosomes, for example, combine liposomes with aninactivated virus. This approach has been shown to result in moreefficient gene transfer in respiratory epithelial cells than eitherviral or liposomal methods alone. Other methods involve mixing otherviral vectors with cationic lipids or hybridising viruses. Each of thesemethods can be used to facilitate transfer of a DNA vector (e.g., aplasmid) into a stem cell of the invention.

Dendrimers, a highly branched macromolecule with a spherical shape, maybe also be used to genetically modify stem cells of the invention. Thesurface of the dendrimer particle may be functionalized to alter itsproperties. In particular, it is possible to construct a cationicdendrimer (i.e., one with a positive surface charge). When in thepresence of genetic material such as a DNA plasmid, chargecomplimentarity leads to a temporary association of the nucleic acidwith the cationic dendrimer. On reaching its destination, thedendrimer-nucleic acid complex can be taken into a stem cell of theinvention by endocytosis.

Toxicology Screening

Stem cells of the invention may also be used in toxicity screening. Forexample, assays can be used to test the potential toxicity of compoundson stem cells of the invention or the differentiated progeny thereof. Inone example, where the stem cells of the invention are differentiatedinto the hematopoietic lineage, hematopoietic stem cells and progenitorassays can be used as to investigate growth and differentiation of cellsin response to positive and negative regulators of hematopoiesis. Theseassays provide the opportunity to assess the potential toxicity ofcompounds on specific hematopoietic (e.g. myeloid, erythroid) cellpopulations.

U.S. Pat. No. 7,166,277, incorporated by reference in its entiretyherein, describes a method of generating neural cells for the purposesof drug screening of putative therapeutic agents targeted at the nervoussystem. Such screening methods can be applied to stem cells of theinvention which have been differentiated into neuronal cell types.

Other approaches include, prior to applying the drug, transforming thecells with a promoter activated by metabolic or toxicologic challengeoperably linked to a reporter gene. Exemplary promoters include thosewhich respond to apoptosis, respond to DNA damage, respond tohyperplasia, respond to oxidative stress, are upregulated in livertoxicity, are responsive to receptors that act in the nucleus,upregulate hepatocyte enzymes for drug metabolism, are from genes whichare deficient in particular disease conditions, and genes which regulatesynthesis, release, metabolism, or reuptake of neurotransmitters. See,for example, the methods and exemplary promoters in U.S. PatentApplication Publication No. 2006/0292695.

In preferred embodiments, for example, stem cell progeny of a selectedcell type can be cultured in vitro can be used for the screening ofpotential therapeutic compositions. These compositions can be applied tocells in culture at varying dosages, and the response of the cellsmonitored for various time periods. Physical characteristics of thecells can be analyzed, for example, by observing cell growth withmicroscopy. The induction of expression of new or increased levels ofproteins such as enzymes, receptors and other cell surface molecules, orother markers of significance (e.g., neurotransmitters, amino acids,neuropeptides and biogenic amines) can be analyzed with any techniqueknown in the art which can identify the alteration of the level of suchmolecules. These techniques include immunohistochemistry usingantibodies against such molecules, or biochemical analysis. Suchbiochemical analysis includes protein assays, enzymatic assays, receptorbinding assays, enzyme-linked immunosorbant assays (ELISA),electrophoretic analysis, analysis with high performance liquidchromatography (HPLC), Western blots, and radioimmune assays (MA).Nucleic acid analysis such as Northern blots can be used to examine thelevels of mR_NA coding for these molecules, or for enzymes whichsynthesize these molecules.

Preservation

Once isolated and/or purified, it may be desirable to preserve the stemcells of the invention. Cell can be preserved by freezing in thepresence of a cryoprotectant, i.e., an agent that reduces or preventsdamage to cells upon freezing. Cryoprotectants include sugars (e.g.,glucose, trehalose), glycols such as glycerol (e.g., 5-20% v/v inculture media), ethylene glycol, and propylene glycol, dextran, anddimethyl sulfoxide (DMSO) (e.g., 5-15% in culture media). Appropriatefreezing conditions (e.g., 1-3° C. per minute) and storage conditions(e.g., between −140 and −180° C. or at −196° C. such as in liquidnitrogen) can be determined by one of skill in the art.

Other preservation methods are described in U.S. Pat. Nos. 5,656,498,5,004,681, 5,192,553, 5,955,257, and 6,461,645. Methods for banking stemcells are described, for example, in U.S. Patent Application PublicationNo. 2003/0215942.

The following examples are intended to illustrate, rather than limit,the invention.

EXAMPLE 1 Purifying And Enriching Stem Cells From Synovial Fluid

In a first step, freshly isolated synovial fluid was obtained from anosteoarthritic (OA) patient. Synovial fluid mononuclear cells were thusderived from joint aspirates. Joint aspirates can be obtained bystandard methods well known to those of skill in the art.

Harvesting SF Stem Cells (Heterogenous Population).

Synovial fluid (SF) from OA patients was harvested, diluted inserum-free medium (AIM-V, GIBCO) or MCDB, MEM, IMDM, RPMI media, andspun at 200 g for 15 minutes at room temperature (RT). The pelletedpopulation was then resuspended in AIM-V up to the original SF volume.The mononuclear cell number from SF ranged between 500,000 to 5 millionheterologous mononuclear cells.

Harvesting SF Stem Cells (Homogenous Population)

The pelleted population was then resuspended in AIM-V, serum free mediumdeveloped for the ex vivo expansion of human lymphocytes, up to theoriginal SF volume, counted, washed, and layered over a discontinuousdensity gradient (ROSETTE SEP DM-M, Stem Cell Technologies). The Rosettediscontinuous density gradient does not allow granulocytes to pelletwith smaller cells, for example, cells smaller than about 6 micrometers(μm) in diameter. In this regard, the gradient is different from, forexample, a FICOLL gradient.

Next, the cells were spun for 500 g (2500 RPM) for 30 minutes at roomtemperature. This characteristic allows only two populations of cells,RBC and stem cells, to pellet, which is useful when enriching for stemcells from bone marrow aspirate. The DM-M separates SF mononuclear cellsinto a buffy layer, which is found at the AIM-V and ficoll interface,and a pelleted layer which is found at the bottom of the conical portionof the tube.

Immunomagnetic Bead Enrichment

Stem cells of the invention do not express major histocompatibilityantigen class I (MHC I) or erythroblast specific glycophorin-A (Gly-A).Therefore, the separated cells were subjected to negative selectionusing anti-class I, CD66b, and anti-Gly-A antibodies. This negativeselection step depletes the population of class I, CD66b, andglycophorin A cells, and recovers the remaining populations, which arefrom about 5% to 30% of the Oct-4 protein expressing cells, and fromabout 1-10% marrow, blood, and tissue mononuclear cells. Cells wereresuspended in blocking buffer and incubated with anti-MHC class I,anti-CD66b, and anti-glycophorin b, immunomagnetic beads for 30 minutesat 4° C. Following incubation, samples were washed and resuspended inmedium at a 10⁵-10⁶ cells per ml and passed through a magnetic field toremove cells bound to immunomagnetic beads.

EXAMPLE 2 Characterization And Expansion Of Enriched Stem Cells

Both populations of separated cells (i.e., from the huffy layer or thepellet) were incubated with anti-MHC class I, anti-CD66b, andanti-glycophorin b, immunomagnetic beads as described above. Sampleswere first assessed for the expression of MEC class I, MHC class II,CD44, CD45, CD13, CD34, CD49c, CD66b, CD73, CD105 and CD90 cell surfacemarkers and then permeabilized to determine the presence of Oct-4,Nanog, Sox-2, Rex-1, GDF-3, and Stella intracellularly. FIGS. 1a-1e aredot plots of the enriched cells, sorted into 3 groups (e.g., Group-A,-B, and -C) and analyzed for Oct-4, Rex-1, Runx2, Sox-9, Nanog, Class I,CD44, and CD45 expression. Six to thirty percent of total mononuclearpopulation, i.e., groups A and B, expressed Oct-4, Nanog, Sox-9, andRex-1 while group C expressed the aforementioned transcription factorsat high levels, with the exception of Oct-4. The majority of the cellswere negative for the above cell surface and intracellular markers, asshown in FIGS. 1a-1e . Evaluation of the mononuclear cell fraction byPCR revealed Nanog, Oct-4, Rex-1, and Sox-2 gene expression (FIG. 2).The molecular weight of the SF transcripts were similar in size to thetranscripts expressed by the Ntera-2 embryonic carcinoma cell line.Accordingly, the SF stem cells express embryonic stem cell genes.

EXAMPLE 3 Propagation Of Synovial Fluid Stem Cells

Heterogenous or homogenous populations of SF stem cells not incubatedwith immunomagnetic beads were plated onto culture dishes coated withabout 7-10 ng/ml serum fibronectin or other appropriate matrix coating.Cells were maintained in Dulbecco Minimal Essential Medium (DMEM) or40-60% (e.g., 60%) Low Glucose DMEM and 40-60% (e.g., 40%) MCDB-201medium supplemented with 1-50 ng/ml (e.g., about 5-15 ng/ml or 10 ng/ml)platelet-derived growth factor-BB (PDGF-BB), 1-50 ng/ml (e.g., about5-15 ng/ml or 10 ng/ml) epidermal growth factor (EGF), 1-50 ng/ml (e.g.,about 5-15 ng/ml or 10 ng/ml) insulin-like growth factor (IGF), or100-10,000 IU (e.g., about 1,000) LW, with 10⁻¹¹ to 10⁻⁸M (e.g., 0.01nM) dexamethasone or other appropriate steroid(s), 2-10 pg/ml (e.g., 4.7ng/ml) linoleic acid, and 0.05-0.15 μm (e.g., 0.1 μm) ascorbic acid. Theculture medium may further include 10-50 ng/ml (e.g., 10 ng/ml) insulin,0-10 ng/ml (e.g., 5.5 ng/ml) transferrin, and 2-10 ng/ml (e.g., 5 ng/ml)selenium. The cells can either be maintained without serum, in thepresence of 1-2% fetal calf serum, or, preferably in 1-2% human AB serumor autologous serum. After 3 days, small colonies of adherent cellsdeveloped, and by days six and nine, the cells became semi-confluent(FIG. 3a ). Freshly isolated SF stem cells were pulsed with CFSE and thepercentage of negative cells was assessed after 6 days. FIG. 3bdemonstrates the proliferation capacity of SF stem cells.

EXAMPLE 4 Formation Of Embryoid Bodies

Stem cells of the invention can form embryoid bodies in culture usingstandard protocols. In one example, EndoCult® Liquid Medium is added toa fibronectin-coated plate (Becton-Dickenson (BD) Biosciences DiscoveryBD Catalog No. 354402). 5×10⁶ cells are plated per well in the 6-wellfibronectin-coated plate and incubated for two days at 37° C., 5% CO₂with >95% humidity. After two days, numerous cell populations includingmature endothelial cells and some monocytes adhere to the bottom of thewell. The non-adherent cells will contain the CFU-Hill colony-formingcells, which are then harvested and further cultured for an additional 3days to allow formation of CFU-Hill colonies. The non-adherent cells arecollected by pipetting the medium in each well up and down vigorously3-4 times to remove any non-adherent cells transiently attached to theadherent population. The non-adherent cells from each well aretransferred into individual 5 ml tubes (BD Catalog No. 352058). Thevolume from each well is measured, e.g., using a 2 ml pipette. Nucleatedcells are counted using 3% acetic acid with Methylene Blue (StemCellTechnologies, Inc., Catalog No. 07060) using a hemacytometer.

Approximately 3.0-3.5×10⁶ cells are expected from one well of a 6-wellplate. From each well, 1×10⁶ cells/well are added to a 24-wellfibronectin-coated plate (BD Catalog No. 354411). Fresh EndoCult® LiquidMedium is added to a final volume of 1.0 ml per well. The cells are thenincubated at 37° C., 5% CO₂ with >95% humidity for three days. Coloniesof cells can then be observed.

EXAMPLE 5 Differentiation

Stem cells of the invention were further characterized to assess theirdifferentiation potential. As an example, after separation from synovialfluid as described in Example 1, the pelleted mononuclear population ofstem cells was cultured in 24-well plates at 10⁵ cell per well. After6-9 days in standard culture medium, lineage-specific differentiationagents were added to culture. The medium was changed every 3-4 days andafter 14 or 21 days, cultures were evaluated. The committed ordifferentiated cells of the invention may be used in the transplantationmethods described above.

Stem cells can also be differentiated using RNAi based methods. In oneexample, trophectoderm production is achieved by transfection of stemcells with OCT-4- or Nanog-targeted RNAi compounds, which reduces thelevels of OCT-4 or Nanog transcripts and proteins. Reduction in OCT-4expression correlated with induction of trophectoderm genes Cdx2, Handl,and PL-1, with formation of cells with trophoblast giant cell phenotypeafter 6 days. Reduction in Nanog expression can correlate with inductionof extraembryonic endoderm genes GATA4, GATA6, and laminin B 1, withsubsequent generation of groups of cells with parietal endodermphenotype. Appropriate RNAi constructs for differentiation into othertissue types can be determined by one of skill in the art.

Differentiation may be carried out by any means known in the art.Exemplary differentiation procedures are described below.

Osteoblast Differentiation

FIG. 4 (left panel) shows an example of osteoblast differentiation.Here, stem cells of the invention were plated into 24-well plates at 10⁵cells per cm² in Cambrex MSC medium for 1 week. On the following day,the medium was replaced with fresh α-MEM containing 10% heat-inactivatedFBS, 1% nonessential amino acids, 1% penicillin and streptomycin, 10 mMa-glycerophosphate, and 50 μM ascorbic acid 2-phosphate, with mediumchanges every 3-4 days. Differentiated stem cells were assayed foralkaline phophatase activity and mineral deposition by histochemicalstaining with the Sigma Kit 85 and Alizarin red methods, respectively,at day 14 (Pittenger et al. (1999) Science; 284:143-147).

Osteoblasts can also be differentiated as follows. Medium from the cellmonolayer is pipetted and discarded. The monolayer is washed with DPBS(Thermo Scientific HyClone ESQualified DPBS, Catalog No. SH30850.03) byadding 10 ml/75 cm² to the flask, being careful not to disturb themonolayer. The flask is rocked back and forth. The DPBS is then removedfrom the monolayer and discarded. Trypsin (Thermo Scientific HyCloneTrypsin, Catalog No. SH30042.01) was added at 3-5 mL/75 cm² flask androcked to cover the monolayer with the trypsin solution. Cells areincubated at 37° C. until the cells begin to detach (approximately 5minutes, but not more than 15 minutes). Complete Mesenchymal Stem CellExpansion Medium 90% Thermo Scientific AdvanceSTEM™ Mesenchymal StemCell Basal Medium, Catalog No. SH30879.02, and 10% Thermo ScientificAdvanceSTEM™ Stem Cell Growth Supplement, Catalog No. SH30878.01) isadded in equal amounts to trypsin, and the cells are pipetted up anddown to form a single cell suspension. The trypsin is removed bycentrifuging the cells at approximately 200 g for 10 minutes at roomtemperature and removing the supernatant. The cell pellet isre-suspended in prewarmed complete Mesenchymal Stem Cell ExpansionMedium at approximately 5 ml/pellet for a 75 cm² flask. A small cellsample is removed and counted with a hemacytometer or cell counter. Thecells are then plated on a fresh tissue culture dish at 80-90%confluency using complete Mesenchymal Stem Cell Expansion Medium. Thecells are allowed to attach for at least 24 hours or until normalmorphology is observed. Once the cells have attached and this level ofconfluency is reached, the medium is removed, the cells are rinsed withtwo rinses of DPBS, and an appropriate amount of complete OsteogenicDifferentiation Medium (90% Thermo Scientific AdvanceSTEM OsteogenicDifferentiation Medium, Catalog No. SH30881.02, and 10% ThermoScientific AdvanceSTEM Stem Cell Growth Supplement, Catalog No.SH30878.02) is added. For a 60 mm dish, about 7 ml is sufficient. Thecells are then incubated at 37° C., 5% CO₂, with humidity. The medium isreplaced every 3 days and osteogenesis typically takes approximately21-28 days. Formation of osteoblasts and mineralized matrix can bedetected by staining protocols known in the art.

In yet another protocol, stem cells are plated at 3,000 cells/cm2 andcultured in the expansion media described above overnight. The followingday, the medium is replaced with fresh α-MEM, 10% pooled human serum(PHS), 1% non-essential amino acids, 1% Pen-Strep, 10 mMβ-glycerophosphate, and 50 μM ascorbic acid 2-phosphate, with mediabeing changed every 3-4 days until osteoblasts form.

Exemplary methods for forming bone are also described in U.S. Pat. No.6,863,900, which describes enhancing bone repair by transplantation ofmesenchymal stem cells. To further enhance bone formation it may bedesirable to inhibit osteoclastogenesis, i.e., cells which decrease bonemass. Such methods are described in U.S. Pat. No. 6,239,157. Stem cellsof the invention may also be used to augment bone formation byadministration in conjunction with a resorbable polymer, e.g., asdescribed in U.S. Pat. No. 6,541,024.

Adipocyte Differentiation

FIG. 4 (middle panel) shows an example of adipocyte differentiation.Here, adipocyte differentiation was induced using a commerciallyavailable adipocyte differentiation kit (Cambrex, East Rutherford, N.J.)according to the manufacturer's recommendations. Differentiated cellswere evaluated by oil red 0 stain. SF stems cell were suspended inMesenchymal Stem Cell Expansion Medium at a density of 100,000 cells perwell in a 24-well culture dish with 1 ml volume per well and incubatedovernight at 37° C. in a 5% CO₂ humidified incubator. When the cellswere 100% confluent, medium was removed from-each well and replaced with0.5-1 ml Adipogenesis Induction Medium (DMEM ˜90%, heat inactivatedfetal bovine serum 10%, 1 μM dexamethasone, 0.5 mM IBMX, 10 μg/mlinsulin, 10% μM indomethacin, 1× penicillin and streptomycin). TheAdipogenesis Induction Medium was replaced every 2-3 days for 21 days.Lipid droplets were detected by microscopic examination as early as 5days into the differentiation period. After 21 days of differentiation,adipocytes were fixed and the lipid droplets stained with Oil Red OSolution.

In another example, adipocyte differentiation may be achieved asfollows. Adipocytes can also be generated using the Thermo ScientificHyClone AdvanceSTEM™ Adipogenic Differentiation Kit. In this procedure,spent media from cultured cells is pipetted from the cell monolayer anddiscarded. The cell monolayer is then washed with Dulbecco's PhosphateBuffered-Saline (Thermo Scientific HyClone ESQualified DPBS Catalog No.SH30850.03) by adding 10 ml/75 cm² to the flask, being careful not todisturb the monolayer. The flask is rocked back and forth, and then theDPBS is removed from the monolayer and discarded. Trypsin (e.g., ThermoScientific HyClone Trypsin (Catalog No. SH30042.01)) is added at 3-5ml/75 cm² flask. The flask is rocked to ensure that the entire monolayeris covered with the trypsin solution, and incubated at 37° C. until thecells begin to detach (about 5 minutes), but no more than 15 minutes.Care should be taken that the cells are not forced to detachprematurely, as this may result in clumping. Next, complete MesenchymalStem Cell Expansion Medium (90% Thermo Scientific AdvanceSTEM™Mesenchymal Stem Cell Basal Medium (Catalog No. SH30879.02) and 10%Thermo Scientific AdvanceSTEM™ Stem Cell Growth Supplement (Catalog No.SH30878.01)) are added in equal amounts to the trypsin solution and thesolution is pipetted up and down until the cells are dispersed into asingle cell suspension. Next, the trypsin is removed by centrifuging thecells at approximately 200 g for 10 minutes at room temperature, and thesupernatant is removed. The cell pellet is then re-suspended inprewarmed complete Mesenchymal Stem Cell Expansion Medium atapproximately 5 ml/pellet for a 75 cm² flask. A small volume sample isthen removed for counting with a hemacytometer or cell counter. Thecells are then plated on a fresh tissue culture dish at 80-90%confluency using complete Mesenchymal Stem Cell Expansion Medium. Thecells are allowed to attach for a minimum of 24 hours, or until normalmorphology is observed. Once the cells have attached and this level ofconfluency is reached, the Mesenchymal Stem Cell Expansion Medium isremoved, the cells are rinsed with two rinses of DPBS, and AdipogenicDifferentiation Medium is added (90% Thermo Scientific AdvanceSTEM™Adipogenic Differentiation Medium (Catalog No. SH30886.02) and 10%Thermo Scientific AdvanceSTEM™ Growth Supplement (Catalog No.SH30878.02)). The amount of medium will vary depending on the size ofthe culture dish being used; for a 60 mm dish, about 7 ml is sufficient.The cells are then incubated 37 C, 5% CO2, with humidity. Every 3 daysthe media is removed and replaced with fresh complete AdipogenicDifferentiation Medium. Adipogenesis can then observed as formation oflipid droplets, generally within 7 days, peaking at 3-4 weeks.

Other commercially available adipocyte kits include Chemicon SCR020.

In another embodiment, adipogenic differentiation is achieved by seedingcells at 10⁴ cells per cm². First, at confluence, cells are put in D10medium and supplemented with 1 μM dexamethasone, 0.2 mM indomethacin, 10μg/ml insulin, and 0.5 mM 3-isobutyl-1-methylxanthine (all fromSigma-Aldrich). Medium is replaced every 3-4 days for 21 days. Cells arewashed three times with PBS, fixed in 10% formalin for 1-2 hours, andstained for 15 minutes with fresh oil red 0 solution (Sigma-Aldrich) todetect adipocyte formation.

Adipocytes can also be differentiated on a solid support, as describedin U.S. Pat. No. 6,709,864.

Neuroectoderm Differentiation

FIG. 4 (right panel) shows one example of ectodermal differentiation.Stem cells were plated in a FN-coated (100 ng/ml) 24-well plate at 3,000or 10⁵ overnight in basal stem cell medium or expansion medium. Thefollowing day, 100 ng/ml bFGF, 10 ng/ml Noggin, 20 μM retinoic acid andcultures continued for 28 days. After 14 days, 10 ng/ml BDNF and GDNFwere also added. Every 7 days, half of the medium was replaced until day28.

Neural differentiation can be evaluated via Q-RT-PCR for early neuraltranscription factors, Islet-1 transcription factor, orthodenticlehomolog 2 (Otx-2), and paired box gene 6 (Pax-6), as well as neural celladhesion molecule and the more mature neuronal marker MAP2, NF200, tau,and myelin basic protein (MBP). Cultures can be further analyzed viaimmunofluorescence for NF200, MAP2, and GFAP.

Neural differentiation can also be achieved as follows. Spent mediumfrom cell monolayer is discarded, and the cells are washed with DPBS(Thermo Scientific HyClone ESQualified DPBS, Catalog No. CSH30850.03) byadding 10 ml/75 cm² to the flask, being careful not to disturb themonolayer. The flask is then rocked back and forth. Next, the DPBS isremoved from the monolayer and discarded. Trysin (Thermo ScientificHyClone Trypsin, Catalog No. SH30042.01) is added at 3-5 ml/75 cm² tothe flask which is rocked to ensure that the entire monolayer is coveredwith the trypsin solution. The flask is incubated at 37° C. until thecells begin to detach (approximately 5, but less than 15, minutes).Complete Thermo Scientific HyClone AdvanceSTEM™ Mesenchymal Stem CellExpansion Media (90% Thermo Scientific AdvanceSTEM™ Mesenchymal StemCell Basal Medium (Catalog No. SH30879.02) and 10% Thermo ScientificAdvanceSTEM™ Stem Cell Growth Supplement (Catalog No. SH30878.01)) isadded in equal amounts to trypsin. The cells are then pipetted up anddown to form a single cell suspension. The trypsin is removed bycentrifuge cells at approximately 200 g for 10 minutes at roomtemperature and aseptically removing the supernatant. The cells areresuspended in prewarmed complete Thermo Scientific HyClone AdvanceSTEM™Neural Differentiation Media (90% Thermo Scientific AdvanceSTEM™ NeuralDifferentiation Medium, Catalog No. SH30893.02 and 10% Thermo Scientific50 ml AdvanceSTEM™ Stem Cell Growth Supplement, Catalog No. SH30878.02)at approximately 5 ml/pellet for a 75 cm² flask. A small sample isremoved for counting with a hemacytometer or cell counter. On a freshtissue culture dish, cells are plated at 30% confluency (approximately2500 cells/cm2) using complete Mesenchymal Stem Cell Expansion Media.The cells are allowed to attach (e.g., for 24 hours or until normalmorphology is seen). Once the cells have attached and this level ofconfluency is reached, the Mesenchymal Stem Cell Expansion Media isremoved. The cells are rinsed with DPBS, and complete NeuralDifferentiation Media is added. For a 60 mm dish, for example, about 7ml is sufficient. The cells are then incubated at 37° C., 5% CO2, withhumidity. Neural differentiation can be observed as formation ofneuron-like cells, typically within 24 hours and peaking at 72 hours. Tomaintain cells in a differentiated state, the media is replaced every 48hours.

Differentiation into neurons, astrocytes, and oligodendrocytes can alsobe achieved as follows. Poly-L-Ornithine coated glass coverslips areplaced into individual wells of a 24-well culture dish (e.g., CorningCatalog No. 3526) containing 1 ml/well of “Complete” NeuroCult® NS-ADifferentiation Medium (Human) (StemCell Technologies. If using BioCoat8-well Culture Slides (pre-coated with Poly-D-Lysine/Laminin, StemCellTechnologies Catalog No. 35-4688, or Poly-D-Lysine Catalog No. 35-4631),add 0.75 ml/well of “Complete” NeuroCult® NS-A Differentiation Medium(Human). Proliferating stem cells are then exchanged in to in “Complete”NeuroCult® NS-A Differentiation Medium (Human) using a 10 ml disposableplastic pipette and centrifuge, and repeat to remove the expansionmedia. The cells are counted using hemacytometer and standard protocols.The cells are then resuspended in an appropriate volume of “Complete”NeuroCult® NS-A Differentiation Medium (Human) to yield a plating celldensity of 0.8-1×10⁵ cells/cm² in 0.75 ml medium on a coated coverslipin a 24-well dish (e.g., 0.9-1.13×10⁵ cells) or in a BioCoat 8-wellCulture Slide (e.g., 0.56-0.7×10⁵ cells). The cells are incubated in a5% CO2 incubator at 37° C. After 5-10 days, the cultures are observedwith an inverted light microscope to determine if cells havedifferentiated (attached) and are viable (phase contrast bright). Platescan be checked daily to determine if the medium needs to be changedduring the differentiation procedure. If the medium becomes acidic(turns yellow), a half medium change is performed. Differentiation canbe assessed using standard methods.

Chondroblast Differentiation

Chondroblast differentiation can be conducted according to methods knownin the art. Such cells may be useful for repair of articular cartilage(e.g., due to injury), in prostheses or in joint (e.g., kneereconstruction), or for cosmetic purposes.

Stem cells of the invention (10⁵ cells per cm²) can be cultured in 1 mlof basal medium (the expansion medium described herein above withoutserum, EGF, or PDGF) with 10 ng/ml TGF-β1 and 100 ng/ml BMP-4 in the tipof a 15-ml conical tube and briefly spun to allow aggregation of thecells in micromass suspension culture. After 9 days, cultures can beevaluated by quantitative reverse-transcription-polymerase chainreaction (Q-RT-PCR) for collagen type II and aggrecan transcripts andstained with Alcian Blue to demonstrate cartilage matrix production.

Chondroblast differentiation can also be conducted as follows. To inducechondrogenic differentiation, spent medium is pipetted from the cellmonolayer and discarded. The monolayer is washed with DPBS (ThermoScientific HyClone ESQualified DPBS (Catalog No. SH30850.03) by adding10 ml/75 cm² to the flask, being careful not to disturb the monolayer.The flask is rocked back and forth. The DPBS is removed and discardedfrom the monolayer. Trypsin (Thermo Scientific HyClone Trypsin (CatalogNo. SH30042.01) is added at 3-5 mL/75 cm² flask and rocked to ensurecoverage of the entire monolayer. The cells are incubated at 37° C.until they begin to detach (5 minutes, but less than 15 minutes). Thetrypsin is then removed by adding an equal volume of completeChondrogenic Differentiation medium (90% Thermo Scientific AdvanceSTEMChondrogenic Differentiation Medium (Catalog No. SH30889.02) and 10%Thermo Scientific AdvanceSTEM Stem Cell Growth Supplement (Catalog No.SH30878.01)), spinning the cells for 10 minutes at 200 g in a swingbucket centrifuge. and gently aspirating the supernatant. Next 4 ml offresh complete Chondrogenic Differentiation medium is added to the tubewithout disturbing the pellet. The cap is then loosely fitted on top ofthe conical tube to allow gas exchange. The tube is then incubated at37° C., 5% CO2, with humidity. The media is replaced every 3 dayswithout disturbing the pellet. Chrondrogenesis generally requires 28days, which can be visualized, for example, by staining and microscopy.

Other methods for differentiation of chondrocytes are described in U.S.Pat. No. 5,908,784. Here, chondrocytes are differentiated culture byculture in a cell pellet, optionally with a corticosteroid such asdexamethasone. Other methods include the use of TGF-β or BMPs such asBMP-2, BMP-12 and BMP-13, with or without ascorbate for chondrocytedifferentiation, as described in U.S. Pat. No. 5,919,702. Any of thesecultures may be performed in three-dimensional culture, as known in theart.

Myocardiocyte Differentiation:

Myocardiocyte differentiation is accomplished by adding basic fibroblastgrowth factor to the standard serum-free culture media without growthfactors. Confluent stem cells are exposed to 5-azacytidine and toretinoic acid and cultured in stem cell expansion medium afterwards.Alternatively, stem cells are cultured with either of these inducersalone or a combination and then cultured in serum-free medium with FGF-2or BMP-4. Cultures are assessed for expression of any of Gata4, Gata6,cardiac troponin-T, cardiac troponin-1, ANP, Myf6 transcription factor,desmin, myogenin, and skeletal actin.

Endothelial Cell Differentiation

Endothelial cell differentiation can be conducted according to methodsknown in the art. Stem cells of the invention can be plated at 0.5-1.010⁵ cells/cm² in basal medium (described above) with 100 ng/ml ofVEGF-165 for 14 days. During the differentiation course, medium can bechanged every 3-4 days. Differentiation cultures can be evaluated byQ-RT-PCR for VWF, CD31/Pecam, fms-like tyrosine kinase-1 (Flt-1), fetalliver kinase-1 (Flk-1), VE-cadherin, tyrosine kinase with Ig, and EGFhomology domains 1 (Tie-1) and tyrosine kinase endothelial (Tek), every3 days until day 10. Differentiated endothelial cells are stained forCD31, VWF, VE-cadherin, and VCAM-1 and evaluated for their ability toform tubes on ECMatrix and uptake acetylated low density lipoprotein(a-LDL). Tube formation can be induced by plating the differentiatedendothelial cells according to the ECM625 angiogenesis assay (Chemicon)per the manufacturer's recommendations, and a-LDL uptake was performedby using Dil-Ac-LDL staining kit (Biomedical Technologies, Stoughton,Mass.) per the manufacturer's recommendations. Briefly, differentiatedstem cells can be incubated with endothelium differentiation mediumcontaining 10 μg/ml Dil-Ac-LDL for 4 hours at 37° C. and rinsed twice byDil-Ac-LDL free endothelium medium. LDL uptake was visualized viafluorescence microscopy.

Hepatocyte Differentiation

Hepatocyte differentiation can be conducted according to methods knownin the art. Hepatocyte differentiation will be achieved by plating0.5-1.0 10⁵ cells/cm² of stem cells on 2% Matrigel-coated (BD354234; BDBiosciences, San Diego) plastic chamber slides in basal medium(described above) with 100 ng/ml FGF-4 and HGF for 15 days. During thedifferentiation course, medium can be changed every 3 days as needed.Differentiation cultures can be evaluated by Q-RT-PCR for HNF-3, HNF-1,CK18 and CK19 albumin, and CYB2B6 every 3 days until day 12.Differentiated cells can be evaluated by immunofluorescence microscopyfor albumin, CK18, and HNF-1 protein expression. To assess the functionof hepatocyte-like cells, karyotyping, telomere length, and telomeraseactivity measurements can be performed. Karyotyping can be conducted byplating enriched cells at 500 cells per cm² 48 hours prior toharvesting, followed by 10 μl/ml colcemid incubation for 2-3 hours.After collection with 0.25% Trypsin-EDTA cells can be lysed with ahypotonic solution and fixation in alcohol. Metaphases can be analyzedafter Giemsa staining. For the telomerase assay, equal numbers ofenriched cells can be lysed in 1× 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) buffer for 10 minutes onice. Debris can then be pelleted at 13,000 g for 10 minutes. Protein canbe quantified by the method of Bradford. One to two μg of protein can beused in the telomere repeat amplification protocol (TRAP). The TRAPprotocol, which uses an enzyme-linked immunosorbent assay (ELISA)-baseddetection system to determine telomerase activity, can be done accordingto the manufacturer's instructions (Chemicon). Positive activity isdefined as OD 450-690 reading >0.2 of test samples after subtractingheat-inactivated controls.

Smooth Muscle Cell Differentiation

Smooth muscle cell differentiation can be conducted according to methodsknown in the art. For example, stem cells of the invention can be platedinto 24-well plates at 3000 or 10⁵ cells/cm² in basal medium (describedabove) supplemented with 10 ng/ml PDGF and 5 ng/ml TGF-β1. During thedifferentiation course, medium can be changed every 3-4 days as needed.Smooth muscle cell (SMC) differentiation can be evaluated by RT-PCR forcalponin, SM actin, smoothelin, gata-6, and myocardin andimmunofluorescence (IF) staining for calponin, SM actin, sm22, andcaldesmon.

Skeletal Muscle Cell Differentiation

Stem cells of the invention can also be differentiated into skeletalmuscle tissue. In one example, 5-Azacytidine can be used todifferentiate stem cells of the invention into muscle cells. Stem cellscan be plated in a variety of densities of 1-4×10⁴ cells per cm² onglass or TPX slides coated with fibronectin, Matrigel, gelatin, orcollagen (Stem Cell Technologies). The cells can then be exposed toconcentrations of 5-azacytidine (e.g., 1-24 μM) for 6-48 hours durationin either 2% human serum, FBS, or serum-free medium (defined as DMEMwith 2 mM L-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycinsupplemented with 10 ng/ml platelet-derived growth factor-BB, andepidermal growth factor (Sigma-Aldrich) and ITS-plus (Fisher ScientificInternational). In some experiments, cells received a further 24-hourexposure to 5-azacytidine 3 days later. Following 5-azacytidineexposure, cells were maintained in serum-free medium for up to 21 days.To augment differentiation after a few days, human serum withdexamethasone and hydrocortisone, myoblast-Conditioned medium, orGalectin-1 may be added.

Myogenic differentiation can be observed by morphological criteria andimmunostaining for desmin and sarcomeric myosin. Myogenic conversion canbe assessed by counting the number of cells positive for desmin andMF20. Pax7, MyoD, and Myogenin expression can be similarly assessedusing immunocytochemical staining.

Pancreatic Islet-Like Differentiation

Stem cell of the invention are plated onto gelatinized dishes in thepresence of LIF, in expansion medium or other appropriate maintenancemedium (e.g., DMEM containing 15% FBS, 1 mM sodium pyruvate, 100 U/mllpenicillin, 100 μg/ml streptomycin, 2 mM glutamine, 0.1 mM Non-essentialamino acids (StemCell Technologies, Catalog No. 07100), 10 ng/ml LW, 100gm MTG). The cells are allowed to grow for two days. Next,Differentiation Medium (15% Fetal Bovine Serum 0.1 mM MEM Non-EssentialAmino Acids (StemCell Technologies, Catalog No. 07600) 2 mM L-Glutamine,and 1 mM MTG in High Glucose DMEM) is added low adherent dishes (e.g.,Ultra-Low Adherent dishes, StemCell Technologies). The stem cells aretrysinized, and resuspended in Differentiation Medium, and plated ontothe low adherent plates. On the second day, the medium is exchanged forfresh Differentiation Medium. The culture continues for 4 days. Next,nestin positive cells are enriched. The cells are transferred to a 14 mlpolystyrene tube, and allowed to settle (3-5 min). The media is removed,and replaced with ES-Cult Basal Medium-A (StemCell Technologies CatalogNo. 07151) supplemented with ITS. The cells are then plated, andcultured for six days, changing media every two days. The medium is thenremoved, the cells are washed with PBS. Cells are then trypsinized, andthe medium is replaced with Pancreatic Proliferation Medium (1× N2Supplement-A (Catalog No. 07152), 1× B27 Supplements 50X (Catalog No.07153), 25 ng/ml recombinant human FGF-b (Catalog No. 02634), andES-Cult™ Basal Medium-A (Catalog No. 05801) to fmal volume of 100 ml).The cells are counted and seeded at 5×105 cells/ml media in a 24 welldish. Media is changed every 2 days for 6 days total. On the sixth day,the medium is replaced with Pancreatic Differentiation Medium (1× N2Supplement-A, 1× B27 Supplements, 10 mM nicotinamide (Catalog No.07154), ES-Cult™ Basal Medium-A to final volume of 100 ml). After sixdays, the insulin production can be detected (e.g., by ELISA).

Other methods for pancreatic cell differentiation can be found in U.S.Pat. No. 6,022,743.

Bone and Bone Cell Production

Stem cells of the invention may also be cultured under conditions whichresult in production of bone or bone cells, and related compositions.Such cells and compositions may be useful, for example, in treating bonediseases such as osteoporosis or to treat injuries to bone.

Bone Marrow Production

Stem cells of the invention may also be used to produce bone marrow orto enhance bone marrow engraftment. Exemplary procedures are describedin U.S. Pat. Nos. 5,733,542 and 5,806,529.

Hematopoietic Stem Cell Production

Stem cells of the invention may also be cultured under conditions thatform hematopoietic stem cells. Exemplary methods for doing so aredescribed in U.S. Patent Application Publication No. 2003/0153082.Briefly, cell can be cultured in the presence of hematogenic cytokinessuch as stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6(IL-6), granulocyte-colony-stimulating factor (G-CSF)—either alone, orin combination with bone morphogenic proteins such as BMP-2, BMP-4, orBMP-7. Typically, at least two, three, or more than three such factorsare combined to create a differentiation cocktail. In one example,embryoid bodies are cultured for 10 days, and then plated in anenvironment containing 100-300 ng/ml of both SCF and Flt-3L, 10-50 ng/mlof IL-3, IL-6, and G-CSF, 100 ng/ml SHH, and 5-100 ng/ml BMP-4—all in amedium containing 20% fetal calf serum or in serum-free mediumcontaining albumin, transferring and insulin. After 8 to 15 days,hematopoietic cells can be evaluated for CD45⁺ and CD34⁺ expression. Inanother example, the cytokines and BMP-4 can be added to the culture thenext day after embryoid body formation, which can further enhance theproportion of CD45⁺ cells after 15 to 22 days. The presence of BMP-4 canallow the user to obtain populations in which 4, 10, or more secondaryCFUs form from each primary CFU, which indicate the presence ofself-renewing hematopoietic progenitors.

Dendritic Cells

Stem cells of the invention may also be cultured under conditions whichform dendritic cells. Such cells may be useful in vaccinations againstcancer by genetically altering the cells to express a cancer antigensuch as telomerase reverse transcriptase (TERT). The vaccine may then beadministered to a subject having a cancer or at increased risk ofdeveloping such a cancer. Exemplary differentiation procedures aredescribed in U.S. Patent Application Publication 2006/0063255. Thus,differentiation can be initiated in a non-specific manner by formingembryoid bodies or culturing with one or more non-specificdifferentiation factors. Embryoid bodies (EBs) can be made in suspensionculture. Undifferentiated stem cells can be harvested by briefcollagenase digestion, dissociated into clusters or strips of cells, andpassaged to non-adherent cell culture plates. The aggregates can be fedevery few days, and then harvested after a suitable period, typically4-8 days. Specific recipes for making EB cells from stem cells of arefound in U.S. Pat. No. 6,602,711, WO 01/51616, and U.S. PatentApplication Publication Nos. 2003/0175954 and 2003/0153082.Alternatively, fairly uniform populations of more mature cells can begenerated on a solid substrate; see, e.g., U.S. Patent ApplicationPublication Nos. 2002/019046.

In one example, the cells can be first differentiated into anintermediate cell (either as an isolated cell type or in situ) that hasfeatures of multipotent hematopoietic precursor cells (e.g.,CD34⁺CD45⁺CD38⁻ and the ability to form colonies in a classic CFUassay). This can be accomplished by culturing with hematopoietic factorssuch as interleukin 3 (IL-3), BMP-4, optionally in combination withfactors such SCF, Flt-3L, G-CSF, other bone morphogenic factors, ormonocyte conditioned medium. The medium used can be any compatiblemedium (e.g., X-VIVO™ 15 expansion medium (Biowhittaker/Cambrex), andAim V (Invitrogen/Gibco)). See also WO 98/30679 and U.S. Pat. No.5,405,772. In addition or as a substitute for some of these factors,hematopoietic differentiation can be promoted by co-culturing with astromal cell lineage (e.g., mouse lines OP9 or Ac-6, commerciallyavailable human mesenchymal stem cells, or the hES derived mesenchymalcell line REF1 (U.S. Pat. No. 6,642,048)), or by culturing mediumpreconditioned in stromal cells culture.

The hematopoietic intermediate can be further differentiated intoantigen presenting cells or dendritic cells that may have one or more ofthe following features in any combination: CD40⁺, CD80⁺, CD83⁺, CD86⁺,Class II MHC⁺, highly Class I MHC⁺, CD14⁻, CCR5⁺, and CCR7⁺. This can beaccomplished by culturing with factors such as GM-CSF, IL-4, or IL-13, apro-inflammatory cytokine such as TNFα or IL-6, and interferon gamma(IFNγ).

Another approach directs stem cells towards the phagocytic or dendriticcell subset early on. Intermediate cells may already bear hallmarks ofmonocytes ontologically related to dendritic cells or phagocytic antigenpresenting cells, and may have markers such as cell surface F4/80 andDec205, or secreted IL-12. They need not have the capability of makingother types of hematopoietic cells. They are made by using IL-3 and/orstromal cell conditioned medium as before, but the GM-CSF is present inthe culture concurrently.

Maturation of the phagocytic or dendritic cell precursor is achieved ina subsequent step: potentially withdrawing the m-3, but maintaining theGM-CSF, and adding IL-4 (or IL-13) and a pro-inflammatory cytokine.Other factors that may be use include IL-1β, IFNγ, prostaglandins (e.g.,PGE2), and transforming growth factor beta (TGFβ); along with TNFαand/or IL-6. A more mature population of dendritic cells can emerge.

In either the above methods, it may be beneficial to mature the cellsfurther by culturing with a ligand or antibody that is a CD40 agonist(U.S. Pat. Nos. 6,171,795 and 6,284,742), or a ligand for a Toll-likereceptor (such as LPS, a TLR4 ligand; poly I:C, a synthetic analog ofdouble stranded RNA, which is a ligand for TLR3; Loxoribine, which aligand for TLR7; or CpG oligonucleotides, synthetic oligonucleotidesthat contain unmethylated CpG dinucleotides in motif contexts, which areligands for TLR9), either as a separate step (shown by the open arrows),or concurrently with other maturation factors (e.g., TNFα and/or IL-6).

In some embodiments, the cells are divided into two populations: one ofwhich is used to form mature dendritic cells that are immunostimulatory,and the other of which is used to form toleragenic dendritic cells. Thetoleragenic cells may be relatively immature cells that are CD80⁻,CD86⁻, and/or ICAM-1⁻. They may also be adapted to enhance theirtoleragenic properties (e.g., transfected to express Fas ligand, orinactivated by irradiation or treatment with mitomycin c).

Functional studies described below can be carried out to characterizethe committed progeny.

Albumin Secretion

Human albumin concentrations can be determined using an ELISA.Concentrations of albumin can be determined by generating standardcurves from known concentrations of human albumin. Peroxidase-conjugatedand affinity-purified anti-human albumin and reference human albumin canbe obtained from Brigham and Women's Hospital Laboratory. To verifyspecificity of results, conditioned medium from endothelialdifferentiations and unconditioned hepatocyte differentiation medium canbe used.

Urea Secretion

Urea secretion can be assessed by colorimetric assay (DIUR-500 BioAssaySystems) per the manufacturer's instructions. Conditioned medium fromendothelial differentiations and unconditioned hepatocytedifferentiation medium can be used as negative controls.

Periodic Acid-Schiff Staining

Slides can be oxidized for 5 minutes in 1% periodic acid-Schiff (PAS)(Sigma-Aldrich) and rinsed several times with double-distilled H2O(ddH2O). Samples can be incubated with Schiff s reagent for 15 minutes,rinsed several times with ddH2O, immediately counterstained withhematoxylin for 1 minute, and washed several times with ddH2O.

The observations made in this example demonstrate that the adultsynovial fluid contains a sub-population of stem cells and that with theappropriate stimuli, these cells can function as mesodermal, ectodermal,or endodermal cell types.

EXAMPLE 4 Isolation Of Proliferative Stem Cells With A Lenti-Oct-4 GfpVector

FACs analysis of the SF stem cells, performed according to the methodsdescribed in Example 2, reveals varying levels of intracellular Oct-4protein expression (FIG. 6). An additional enrichment scheme involvesthe use of a vector to isolate a proliferative stem cell of theinvention. The vector comprises a stem cell-specific promoter coupled toa heterologous nucleic acid sequence encoding at least one selectablemarker gene, which enables isolation of the desired stem cell.

After separation from synovial fluid as described in Example 1, thepelleted mononuclear population of stem cells were resuspended to 10⁵per ml and placed in 6-well tissue culture plastic. Lenti-Oct-4-GFP wasadded to the suspension and cultured for three days (FIG. 7a ), fourdays (FIG. 7b ), and nine days (FIG. 7c ). The GFP-expressing cells weresubsequently sorted into individual wells by flow cytometry. FIG. 6shows a slide dot plot of the Oct-4 intracellular stain of the pelletedpopulation of stem cells. 30% of the freshly isolated synovial fluidstem cells are Oct-4+ but in most samples, Oct-4 is expressed in 5%-6%of the stem cells.

Vector Design

FIGS. 8a-8c show a vector map of an exemplary lentiviral vector for usein the invention. The lentiviral vector shown in panel a is used for thestem cell specific expression of H2B-EGFP in stem cells. The lentiviralvector in panel b is used for the stem cell specific expression ofGFP-ZEOCIN in stem cells. In certain embodiments of the invention, thevector may be used for the stem-cell specific expression of a masterregulator gene, for example as shown in FIG. 6c , where the lentiviralvector is constructed for the stem-cell specific expression of IRES EGFPin stem cells.

In other embodiments of the invention, co-transducible viral vectors aredesirable for the tetracycline-inducible and stem/lineage progenitorcell-specific expression of a master regulator gene, for example CDX4and/or one of the HOX genes, and IRES EGFP in stem cells. FIGS. 9a and9b show a cotransducible lentiviral vector that is suitable for useaccording to this embodiment.

Transduction and Selection

FIGS. 10a and 10b show schematics of lentiviral constructs containing astem cell-specific promoter, for example Oct-4, Nanog, HTert, Rex, thatis capable of driving the expression of a marker such as H2B-EGFP or GFPin stem cells. Following transduction with the lentivirus, the cells areleft in culture for 24-72 hours. At this time, selection is carried out.A number of methods are useful for selection, dependent upon theconstruction of the vector. For instance, FACS sorting can be used tosort EGFP⁺ cells. Alternatively, blasticidin or Zeocin selection can beused when appropriate. Accordingly, following selection those transducedcells expressing H2B-EGFP will be sorted and selected. FIG. 11 a showsthe same experimental procedure, using a lentiviral vector thatexpresses a master regulator gene, for example CDX4 or a HOX gene. FIG.11b shows cotransduction of a lentiviral vector that uses a stem cellpromoter driving the expression of a tetracycline (TA)-IRES-EGFPtogether with a lentiviral vector containing tetracycline (tetO) linkedto CMV promoter (CMVmin) and a master gene. The experiments can also beapplied in vivo. FIG. 12a shows the generation of humanized rTtAtransgenic mice for the in vivo expansion of repopulating human celllineage-specific progenitor/stem cells. Transgenic mice comprising rTtAthat is knocked in downstream of a cell lineage promoter, for exampleVav promoter, for expression in mouse stem cells. These mice are crossedwith transgenic mice comprising tetracycline responsive promoter drivingthe expression of diphtheria toxin, for example tetO-CMVmin-DTA. Thiscross produces a transgenic mouse for tetracycline ablation of mousecell-specific lineages. Addition of doxycycline allows for selection oftetracycline-resistant cells. FIG. 12b shows the generation of humanizedrTtA transgenic mice for the in vivo expansion of repopulating humancell lineage-specific progenitor/stem cells (e.g. human CD34⁺repopulating hematopoietic stem cells).

Lentiviral transduction can be carried out using the VIRAPOWER T-REXLentiviral Expression System, a product of Invitrogen (full productinformation available on the world wide web atinvitrogen.com/content/sfs/manuals/virapowertrex_lenti_man.pdp. TheVIRAPOWER T-REX Lentiviral Expression System is a Gateway-adapted,lentiviral destination vector for high-level, regulated expression individing and non-dividing mammalian cells. The VIRAPOWER LentiviralTechnology facilitates highly efficient, in vitro or in vivo delivery ofa target gene or RNA to dividing and non-dividing mammalian cells usinga replication-incompetent lentivirus. The TREX Technology facilitatestetracycline-regulated expression of a gene of interest in mammaliancells through the use of regulatory elements from the E. coliTn10-encoded tetracycline (Tet) resistance operon (Hillen and Berens,Annu. Rev. Microbiol. 48, 345-369, 1994; Hillen et al., J. Mol. Biol.169, 707-721, 1983). Tetracycline regulation in the T-REX System isbased on the binding of tetracycline to the Tet repressor andderepression of the promoter controlling expression of the gene ofinterest (Yao et al., Hum. Gene Ther. 9, 1939-1950, 1998). When theinducible expression construct and the regulatory expression constructare present in the same mammalian cell, expression of the gene ofinterest is repressed in the absence of tetracycline and induced in itspresence (Yao et al., as above). GATEWAY Technology is a universalcloning method that takes advantage of the site-specific recombinationproperties of bacteriophage lambda (Landy, 1989) to provide a rapid andhighly efficient way to move the DNA sequence of interest into multiplevector systems. The expression system contains the gene of interestunder the control of a tetracycline-regulatable, hybrid CMV/TO promoter.This expression plasmid contains elements that allow packaging of theconstruct into virions and the ZEOCIN resistance marker for selection ofstably transduced cell lines. The system includes an expression plasmidthat constitutively expresses high levels of the tetracycline (Tet)repressor under the control of a CMV promoter. This expression plasmidalso contains elements that allow packaging of the construct intovirions and the Blasticidin resistance marker for selection of stablytransduced VIRAPOWER T-REX cell lines.

Those skilled in the art will recognize, or be able to determine usingno more than routine experimentation, variations on the foregoingexamples that will permit them to identify many other stem cells thanthe ones described herein.

Other Embodiments

All patents, patent applications, and publications mentioned in thisspecification including U.S. Provisional Application No. 60/927,596,filed May 3, 2007 are herein incorporated by reference to the sameextent as if each independent patent, patent application, or publicationwas specifically and individually indicated to be incorporated byreference.

The invention claimed is:
 1. A method of enhancing recovery from jointsurgery, comprising administering to the joint during surgery to repairthe joint a composition comprising stem or progenitor cells that expressa stem cell transcription factor but do not detectably express MHC ClassI or cell surface markers CD13, CD44, CD45, and CD90 in an amounteffective to enhance recovery, and a pharmaceutically acceptablecarrier.
 2. The method of claim 1, wherein the stem cells are capable ofproliferating and differentiating into ectoderm, mesoderm and endoderm.3. The method of claim 1, wherein the stem cells are synovial fluidderived, blood derived or tissue derived.
 4. The method of claim 1,wherein the stem cells are isolated from a mammal.
 5. The method ofclaim 4, wherein the stem cells are isolated from a human.
 6. The methodof claim 4, wherein the stem cells are isolated from an adult mammal. 7.The method of claim 1, wherein the stem cells are autologous withrespect to a recipient undergoing the joint surgery.
 8. The method ofclaim 1, wherein the stem cells are allogeneic with respect to arecipient undergoing the joint surgery.
 9. The method of claim 1,wherein the stem cells are committed to differentiate.
 10. The method ofclaim 1, wherein the composition further comprises at least onesubpopulation of differentiated progeny cells.
 11. The method of claim1, wherein the population of cells expresses a stem cell transcriptionfactor selected from the group consisting of Oct-4, Nanog, Sox-2, Rex-1,GDF-3 and Stella.